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Advances in Enzymology and Related Areas of Molecular Biology, Volume 41
Abstract
IntroductionTritiation of AsialoglycoproteinsSpecificity of GalactoseLocus of Uptake and CatabolismEnzymatic Replacement of Sialic Acid ResiduesQuantitation of the Galactose RequirementGenerality of the PhenomenonDifferentiation from PhagocytosisMechanism of Hormone InactivationChemical Coupling of Glycopeptides to ProteinsNew Method of Labeling GlycoproteinsBinding by Plasma MembranesQuantitation of BindingSolubilization of Binding ProteinSummary
... Sialic acids induce immune tolerance, inhibit immune cells and regulate immune cell migration [15,17]. The terminally bound sialic acids are of particular importance for fetuin-A metabolism as they protect circulating fetuin-A from hepatic clearance through the asialoglycoprotein receptor, which is expressed on hepatocytes and liver macrophages [18,19]. Attenuated fetuin-A sialylation has been observed in autoimmune diseases [17], in neonates with intrauterine growth retardation [20] and in patients with rheumatoid arthritis [21], whereas increased sialylated fetuin-A serum levels were recorded in patients with allergies [22]. ...
... This has been reported for spinal cord injuries and other pathologic conditions [65]. Second, sialidase activity in inflammation will produce asialofetuin-A, which is readily cleared by the hepatic asialoglycoprotein receptor (for details see above) [16,18,19,62]. Accordingly, we showed increased asialofetuin-A in neuroinflammation. ...
... It also makes an intrathecal synthesis of fetuin-A unlikely as this might lead to different sialylation patterns of fetuin-A in serum and CSF. As already mentioned above, asialofetuin-A is rapidly cleared from circulation after binding to the hepatic asialoglycoprotein receptor [18,19]. Among the here studied samples, double bands, i.e. asialofetuin-A, were predominantly detected in the context of inflammatory CNS disorders. ...
Fetuin-A is a liver derived plasma protein showing highest serum concentrations in utero, preterm infants, and neonates. Fetuin-A is also present in cerebrospinal fluid (CSF). The origin of CSF fetuin-A, blood-derived via the blood-CSF barrier or synthesized intrathecally, is presently unclear. Fetuin-A prevents ectopic calcification by stabilizing calcium and phosphate as colloidal calciprotein particles mediating their transport and clearance. Thus, fetuin-A plays a suppressive role in inflammation. Fetuin-A is a negative acute-phase protein under investigation as a biomarker for multiple sclerosis (MS). Here we studied the association of pediatric inflammatory CNS diseases with fetuin-A glycosylation and phosphorylation. Paired blood and CSF samples from 66 children were included in the study. Concentration measurements were performed using a commercial human fetuin-A/AHSG ELISA. Of 60 pairs, 23 pairs were analyzed by SDS-PAGE following glycosidase digestion with PNGase-F and Sialidase-AU. Phosphorylation was analyzed in 43 pairs by Phos-Tag TM acrylamide electrophoresis following alkaline phosphatase digestion. Mean serum and CSF fetuin-A levels were 0.30 ± 0.06 mg/ml and 0.644 ± 0.55 μg/ml, respectively. This study showed that serum fetuin-A levels decreased in inflammation corroborating its role as a negative acute-phase protein. Blood-CSF barrier disruption was associated with elevated fetuin-A in CSF. A strong positive correlation was found between the CSF fetuin-A/serum fetuin-A quotient and the CSF albumin/serum albumin quotient, suggesting predominantly transport across the blood-CSF barrier rather than intrathecal fetuin-A synthesis. Sialidase digestion showed increased asialofetuin-A levels in serum and CSF samples from children with neuroinflammatory diseases. Desialylation enhanced hepatic fetuin-A clearance via the asialoglycoprotein receptor thus rapidly reducing serum levels during inflammation. Phosphorylation of fetuin-A was more abundant in serum samples than in CSF, suggesting that phosphorylation may regulate fetuin-A influx into the CNS. These results may help establish Fetuin-A as a potential biomarker for neuroinflammatory diseases.
... There are striking similarities between the substances active in our system and those which are taken up by the liver asialoglycoprotein receptor (26,(33)(34)(35)(36); here the most active ligands also terminate in Gal or GalNAc, are clustered, and Ca% a prerequisite for activity (33,34,50). However, at 4 "C the rate of binding of asialo-orosomucoid to asialoglycoprotein receptor was rapid (26,33,51) whereas adhesion of ESb cells to hepatocytes was minimal (29). ...
... There are striking similarities between the substances active in our system and those which are taken up by the liver asialoglycoprotein receptor (26,(33)(34)(35)(36); here the most active ligands also terminate in Gal or GalNAc, are clustered, and Ca% a prerequisite for activity (33,34,50). However, at 4 "C the rate of binding of asialo-orosomucoid to asialoglycoprotein receptor was rapid (26,33,51) whereas adhesion of ESb cells to hepatocytes was minimal (29). ...
... There are striking similarities between the substances active in our system and those which are taken up by the liver asialoglycoprotein receptor (26,(33)(34)(35)(36); here the most active ligands also terminate in Gal or GalNAc, are clustered, and Ca% a prerequisite for activity (33,34,50). However, at 4 "C the rate of binding of asialo-orosomucoid to asialoglycoprotein receptor was rapid (26,33,51) whereas adhesion of ESb cells to hepatocytes was minimal (29). ...
... 51,53−60 ASGPR shows high affinity and specificity toward multiantennary galactose and N-acetyl galactosamine (GalNAc) and induces receptor-mediated endocytosis. 61,62 Whereas the first reported targeted gene delivery focused on natural ligands for targeting ASGPR, 63,64 artificial GalNAc ligands became extensively studied and were continuously optimized for improved nucleic acid transfer to hepatocytes. 60,65 This optimization resulted in the market approval of several direct ligand−siRNA conjugates for the treatment of rare diseases. ...
A cationic, dendrimer-like oligo(aminoamide) carrier with four-arm topology based on succinoyl tetraethylene pentamine and histidines, cysteines, and N-terminal azido-lysines was screened for plasmid DNA delivery on various cell lines. The incorporated azides allow modification with various shielding agents of different polyethylene glycol (PEG) lengths and/or different ligands by copper-free click reaction, either before or after polyplex formation. Prefunctionalization was found to be advantageous over postfunctionalization in terms of nanoparticle formation, stability, and efficacy. A length of 24 ethylene oxide repetition units and prefunctionalization of ≥50% of azides per carrier promoted optimal polyplex shielding. PEG shielding resulted in drastically reduced DNA transfer, which could be successfully restored by active lectin targeting via novel GalNAc or mannose ligands, enabling enhanced receptor-mediated endocytosis of the carrier system. The involvement of the asialoglycoprotein receptor (ASGPR) in the uptake of GalNAc-functionalized polyplexes was confirmed in the ASGPR-positive hepatocarcinoma cell lines HepG2 and Huh7. Mannose-modified polyplexes showed superior cellular uptake and transfection efficacy compared to unmodified and shielded polyplexes in mannose-receptor-expressing dendritic cell-like DC2.4 cells.
... Platelets with reduced levels of α2,3-linked sialic acids expose β-gal and are cleared through the Ashwell-Morell receptor (AMR), which is expressed on hepatocytes [294]. The AMR exists as a heterooligomeric receptor with two subunits, asialoglycoprotein receptors 1 and 2 (ASGR1/2) [295,296]. ...
There is accumulating evidence that platelets play roles beyond their traditional functions in thrombosis and hemostasis, e.g., in inflammatory processes, infection and cancer, and that they interact, stimulate and regulate cells of the innate immune system such as neutrophils, monocytes and macrophages. In this review, we will focus on platelet activation in hemostatic and inflammatory processes, as well as platelet interactions with neutrophils and monocytes/macrophages. We take a closer look at the contributions of major platelet receptors GPIb, αIIbβ3, TLT-1, CLEC-2 and Toll-like receptors (TLRs) as well as secretions from platelet granules on platelet–neutrophil aggregate and neutrophil extracellular trap (NET) formation in atherosclerosis, transfusion-related acute lung injury (TRALI) and COVID-19. Further, we will address platelet–monocyte and macrophage interactions during cancer metastasis, infection, sepsis and platelet clearance.
... EUL: Euonymus europaeus lectins; Nictaba: Nicotiana tabacum agglutinin; ABA: Agaricus bisporus agglutinin; CRA: Chitinase V related agglutinin; GNA: Galanthus nivalis agglutinin; F-type: fucose-binding fold containing; C-type: calcium-dependent; PA14-like: PA14 domain containing proteins; Ptype: mannose-6-phosphate receptors; and X-type: Xenopus laevis oocyte cortical granule lectin-like. Morell, 1974). Later, many lectins were identified in bacteria, fungi, protozoans, invertebrates, and vertebrates (Kilpatrick, 2002;Nizet et al., 2017). ...
Lectins are ubiquitous proteins that reversibly bind to specific carbohydrates and, thus, serve as readers of the sugar code. In photosynthetic organisms, lectin family proteins play important roles in capturing and releasing photosynthates via an endogenous lectin cycle. Often, lectin proteins consist of one or more lectin domains in combination with other types of domains. This structural diversity of lectins is the basis for their current classification, which is consistent with their diverse functions in cell signaling associated with growth and development, as well as the response of plants to biotic, symbiotic, and abiotic stimuli. Furthermore, the lectin family shows evolutionary expansion that has distinct clade-specific signatures. Although the function(s) of many plant lectin family genes are unknown, studies in the model plant Arabidopsis thaliana have provided insights into their diverse roles. Here, we used a biocuration approach rooted in the critical review of scientific literature and information available in the public genomic databases to summarize the expression, localization, and known functions of lectins in Arabidopsis. A better understanding of the structure and function of lectins will add to improvements of agricultural productivity through the manipulation of candidate genes for breeding climate-resilient crops, or by regulating metabolic pathways by applications of plant growth regulators.
... However, due to its potential pathophysiological relevance it was included herein. does not seem to increase the clearance of Tf [208], and desialylated isoforms can be identified in the blood plasma [74,209]. A specific asialo-transferrin isoform is expressed in the brain, lacking the terminal sialyl-galatose moiety in its carbohydrate branches [210]. ...
Transferrin (Tf) is an essential protein, probably ubiquitous to all metazoans. The main function of this family of proteins is to bind and transport ferric ions, increasing Fe(III) solubility under physiological conditions and preventing its deleterious pro-oxidant role in aerobic environments. In humans, Tf is responsible for the safe transport of this essential micronutrient through circulation and its delivery to requiring cells. Cellular uptake occurs through endocytosis mediated by the transferrin receptor (TfR). Although there is a good understanding of the general molecular mechanisms governing Tf iron binding and cellular iron delivery, several aspects of Tf biochemistry remain unclear.
In this review, we provide an overview of the inorganic biochemistry of human Tf (hTf), exploring the available structural information on hTf and the hTf/TfR complex to discuss physiologically relevant aspects, such as iron binding site distribution, mechanism of iron loading and TfR priming of iron release in vivo. In addition, we consider the role of hTf microheterogeneity on its function. Post-translational modifications such as phosphorylation, glycation and oxidation may occur at relevant hTf sites, which will compromise iron binding, inter lobe cooperativity or interaction with the TfR. Furthermore, these modifications may contribute to the deregulation of systemic iron transport and distribution on a disease specific manner. Finally, a brief review of the role of hTf as one of the main blood serum ligands for toxic and therapeutic metal ions is presented.
hTf is a long known and studied protein, but it is essential to understand the factors governing its expression and catabolism and how specific modifications modulate its function, in order to fully comprehend its role in human pathology or to explore its potential as a therapeutic agent.
... Therefore, we isolated circulating EVs from serum using qEV columns ( Figure 3A) and detected exercise-induced metabolic proteins related to skeletal muscle, adipose, and liver in representative six subjects. We detected CD9, CD81, ALIX, TSG101, syntenin-1, annexin V (EV markers), α-skeletal muscle actin (α-skeletal MA-enriched in skeletal muscle) (Ilkovski et al., 2005), ASGPR1 (enriched in hepatocytes) (Ashwell and Morell, 1974;Povero et al., 2014), and perilipin A (enriched mainly in the adipose tissue) (Kimmel et al., 2010;Eguchi et al., 2016) in circulating EVs taken at T 0 ( Figure 3B and Supplementary Figure 1), suggesting that a part of circulating EVs were derived from multiple tissues and cells including the skeletal muscle, hepatocytes, and adipose tissue as a function of HIIT-Ex. Although we used qEV column that has less lipoprotein overlap for EV isolation, a small amount of apolipoprotein A1 was still detected as a contamination in all circulating EVs (Figure 3B and Supplementary Figure 1). ...
Introduction/Purpose
High-intensity interval training (HIIT) promotes various biological processes and metabolic effects in multiple organs, but the role of extracellular vesicles (EVs) released from a variety of cells is not fully understood during HIIT exercise (HIIT-Ex). We investigated the changes in circulating number and proteomic profile of EVs to assess the effect of HIIT-Ex.
Methods
Seventeen young men (median age, 20 years) were enrolled in the study. Total duration of the HIIT-Ex was 4 min. Blood samples were collected from before HIIT-Ex (pre-HIIT-Ex), at the immediate conclusion of HIIT-Ex (T 0 ), at 30 min (T 30 ), and at 120 min after HIIT-Ex. The pulse rate and systolic blood pressure were measured. Circulating EVs were characterized, and EV proteins were detected via nano liquid chromatography tandem mass spectrometry.
Results
The pulse rate and systolic blood pressure at T 0 to pre-HIIT-Ex were significantly higher. Circulating EV number was significantly altered throughout the HIIT-Ex, and the source of circulating EVs included skeletal muscle, hepatocytes, and adipose tissue. Proteomic analysis identified a total of 558 proteins within isolated circulating EVs from pre-HIIT-Ex, T 0 , and T 30 . Twenty proteins in total were significantly changed at pre-HIIT-Ex, T 0 , and T 30 and are involved in a variety of pathways, such as activation of coagulation cascades, cellular oxidant detoxification, and correction of acid–base imbalance. Catalase and peroxiredoxin II were increased at T 0 .
Conclusion
The circulating EV composition can be immediately changed by particularly a short time of HIIT-Ex, indicating that EVs may intercommunicate across various organs rapidly in response to HIIT-Ex.
Background and aims
Current research has suggested that asialoglycoprotein receptor 1 (ASGR1) is involved in cholesterol metabolism and is also related to systemic inflammation. This study aimed to assess the correlation between the serum soluble ASGR1 (sASGR1) concentration and inflammatory marker levels. Moreover, the second objective of the study was to assess the association between sASGR1 levels and the presence of coronary artery disease (CAD).
Methods
The study subjects included 160 patients who underwent coronary angiography. Ninety patients were diagnosed with CAD, while seventy age- and sex-matched non-CAD patients served as controls. We measured the serum sASGR1 levels using an ELISA kit after collecting clinical baseline characteristics.
Results
Patients with CAD had higher serum sASGR1 levels than non-CAD patients did (P < 0.0001). sASGR1 was independently correlated with the risk of CAD after adjusting for confounding variables (OR = 1.522, P = 0.012). The receiver operating characteristic (ROC) curve showed that sASGR1 had a larger area under the curve (AUC) than did the conventional biomarkers apolipoprotein B (APO-B) and low-density lipoprotein cholesterol (LDL-C). In addition, multivariate linear regression models revealed that sASGR1 is independently and positively correlated with high-sensitivity C-reactive protein (CRP) (β = 0.86, P < 0.001) and WBC (β = 0.13, P = 0.004) counts even after adjusting for lipid parameters. According to our subgroup analysis, this relationship existed only for CAD patients.
Conclusion
Our research demonstrated the link between CAD and sASGR1 levels, suggesting that sASGR1 may be an independent risk factor for CAD. In addition, this study provides a reference for revealing the potential role of sASGR1 in the inflammation of atherosclerosis.
The property of lectins to specifically recognize and bind carbohydrates makes them an excellent candidate in biomedical research. Among them are fucose-binding lectins possessing the capacity to bind fucose are taxonomically, evolutionarily and ecologically significant class of lectins that are identified in a wide range of taxa. Purification of fucose-binding lectins dates back to 1967 when L-fucose binding protein from Lotus tetragonolobus was isolated using a dye that contained three α-L-fucopyranosyl residues. Beginning with that, several FBLs were purified from various animals as well as plant sources that were structurally and functionally characterised. This review focuses on fucose-binding lectins, their occurrence and purification with special emphasis on various strategies adopted to purify them followed by molecular and functional characterization. The exclusive ability to recognize and bind to fucose-containing glycans endows these lectins
with the potential to act as anti-cancer agents, diagnostic markers and mitogens for immune cells. Though they have been in research focus for more than half a century with their occurrence reported in various taxa, they still need to be explored
for their prospective functions to develop them as a biological tool in biomedical research.
Nucleic acid therapeutics have shown great potential for the treatment of numerous diseases, such as genetic disorders, cancer and infections. Moreover, they have been successfully used as vaccines during the COVID-19 pandemic. In order to unfold full therapeutical potential, these nano agents have to overcome several barriers. Therefore, directed transport to specific tissues and cell types remains a central challenge to receive carrier systems with enhanced efficiency and desired biodistribution profiles. Active targeting strategies include receptor-targeting, mediating cellular uptake based on ligand-receptor interactions, and chemical targeting, enabling cell-specific delivery as a consequence of chemically and structurally modified carriers. With a focus on synthetic delivery systems including polyplexes, lipid-based systems such as lipoplexes and lipid nanoparticles, and direct conjugates optimized for various types of nucleic acids (DNA, mRNA, siRNA, miRNA, oligonucleotides), we highlight recent achievements, exemplified by several nucleic acid drugs on the market, and discuss challenges for targeted delivery to different organs such as brain, eye, liver, lung, spleen and muscle in vivo.
В обзоре представлены современные данные о структуре и функциональной роли молекул клеточной адгезии, принадлежащих к семейству селектинов — селектинов Р, L и Е, — и их участии в патогенезе сердечно-сосудистых заболеваний. Молекулы межклеточной адгезии эндотелия сосудистой стенки, тромбоцитов и лейкоцитов служат важным звеном в процессах васкулогенеза, развития и регенерации сосудистой системы, с одной стороны, и участниками наиболее ранних этапов нарушения функции эндотелия с последующим развитием патологии — с другой. По этой причине для понимания молекулярных основ патогенеза сердечно-сосудистых заболеваний очень важны представления о механизмах деятельности данной группы молекул. Адгезия молекул, как между клетками, так и между клетками и компонентом внеклеточного матрикса, — важнейший этап физиологических и биохимических процессов. На сегодняшний день известно пять классов молекул межклеточной адгезии: интегрины, кадгерины, иммуноглобулины (в том числе нектины), селектины и адрессины. Все они связаны с цитоплазматической мембраной и обеспечивают взаимодействие клеток друг с другом. Некоторые из них являются трансмембранными и связаны с цитоскелетом клетки. На поверхности клеток молекулы межклеточной адгезии могут располагаться кластерами, образуя участки многоточечного связывания и тем самым определяя степень авидности. Одна из наиболее значимых функций селектинов — участие в начальной стадии каскада адгезии лейкоцитов, в результате которой происходят их связывание с эндотелием, роллинг и дальнейшая экстравазация в ткани. Первый этап этого процесса опосредован специфическими нековалентными взаимодействиями между селектинами и их гликановыми лигандами, при этом гликаны функционируют в качестве интерфейса между лейкоцитами или раковыми клетками и эндотелием. Нацеленность на эти взаимодействия остаётся одной из основных стратегий, направленных на разработку новых методов лечения иммунных, воспалительных и онкологических заболеваний.
Lectins are a class of specific carbohydrate binding proteins that can agglutinate cells or precipitate polysaccharides and glycoproteins and function as recognition molecules. They vary in molecular size, amino acid composition, three-dimensional structure, metal requirement, and function in order to adapt to the challenges of the antigens/ pathogens in the surrounding environment. There are different types of lectins such as C-type (calcium dependent lectin), S-type or Galectins (β-galactoside binding lectins), L-type (lectins containing leguminous lectin domain), MBL (mannose-binding lectin), P-type (mannose 6-phosphate receptor specific lectin), N-type (recognize mannose 6-phosphate N-glycans), R-type (contain structurally similar CRD to ricin), Collectins (collagen-containing C-type lectins), I-type (immunoglobulin type lectin), etc. Siglecs are a group of I-type lectins with sialic acid (Sia)-specificity and an amino-terminal. Such sialic acid specific lectins are of growing interest as reagents in biochemical research and diagnostic analysis and can be extremely useful to study sialic acids in diagnosis of pathogenic disease and tumors. The prevalence of such impressive sialic acid specific lectin with significant biomedical prospective is reported among arthropods and molluscs. Among these, the lectins of crabs were studied elaborately and almost all reported crab lectins, especially brachyuran crabs are of sialic acid specific. This chapter deliberates the lectins identified from anomuran and brachyuran crabs.
Host–pathogen interactions are complex, governed by numerous factors such as host’s age, environment, behavior, immune system, and characteristics of the pathogen. The outcome of such a relationship depends on the resistance and susceptibility of the immune reactions of host. In fishes, there are two type immune responses, adaptive and innate. One of the key components of innate immune response is lectins. Lectins attach to the carbohydrate component of the glyco-proteins or lipids, proteoglycans, and are basically proteins. They not only mediate protein–carbohydrate interactions for the identification of infectious agents but also involved in biological activities of immune system like immobilization, agglutination, opsonization, and inactivation of pathogens. Studies on identification and characterization of lectins in fish are essential for prophylaxis, tracking, and treatment of diseases.KeywordsHost–pathogenImmune systemFish lectins
Lectins are carbohydrate-binding proteins found in viruses, prokaryotes, and eukaryotes. Lectins are associated with numerous biological capacities like cell attachment, phagocytosis, complement activation, and innate immunity. Fish lectins play a significant part in treatment, embryogenesis, and morphogenesis. There are many different types of fish lectins, and they are classified based on their structure, function, namely galectins, C-type, Pentraxins, Calnexins, I-type, F-type, and l-rhamnose lectins. The molecular cloning of different fish lectin-associated genes and its promoter regions has been cloned over past years. The cloning of cDNA sequences by techniques including RACE-PCR yields full-length cDNA. Also, for the cloning of fish lectins, gene-specific primer pairs were developed which was based on the expressed sequence tag (EST) acquired from random sequencing of a cDNA library utilizing fish liver and other organs. Fish lectins were successfully cloned, and its potential function in fish immunology was also investigated according to the literature. The CRISPR/Cas9 system is a strong, effective tool for editing genomic DNA sequences, including gene knockout in fish, using RNA-guided site-specific DNA cleavage. The efficacy of CRISPR/Cas9 knockdown via microinjection was demonstrated utilizing two genes in fish lectins such as TICAM-1 and RBL. CRISPR technology was found to be an efficient way to expedite genetic improvement of farmed aquatic fishes and can be depicted as a promising strategy for the efficiency, precision, and predictability in aquaculture research. These techniques might contribute to a better understanding of the specific fish lectins involved in host–pathogen relationship contributing to fish innate immunity and can aid in exploring novel lectins or cloning procedures for aquaculture development.KeywordsFish lectinMolecular cloningMolecular interactionCRISPR
Lectins are proteins that can bind to carbohydrates selectively and reversibly without changing the ligand’s covalent structure. Lectins are divided into several families based on their structure, binding specificities, and calcium dependence, such as C-type lectins, I-type lectins, F-type lectins, intelectins, rhamnose binding lectins, galectins, Lily-type lectins, and so on. Innate immunity and disease resistance are known to be aided by lectins such as ficolins, calnexin, galectins, F-type lectins, intelectins, and mannose-binding proteins (MBPs). In fish, skin serves as an essential immunological structure, and lectins have been found in the stomach, eggs, gut, liver, gills, serum, skin, and plasma of several fish species. The capacity of lectins to promote agglutination, suppression of planktonic development, biofilm inhibition or eradication, and/or death of bacteria has been shown to have direct antibacterial action. The interaction of lectins with bacterial cell wall components [such as N-acetylglucosamine (NAG), N-acetylmuramic acid (NAM), tetrapeptides related to NAM, and lipopolysaccharides] as well as membrane receptors has been credited with growth suppression and death induction. This might cause permeabilisation and the development of holes in the bacterial cell wall and membrane, allowing intracellular material to seep out. Lectins are part of the innate immune system’s humoral component and are engaged in the detection of PAMPs that cause agglutination and neutralisation of potentially microbial pathogens or the activation of complement components. Various research findings show that fish lectins play an important role in immunological identification of microbial infections and phagocytosis clearance. The significance of lectins in antimicrobial and immunomodulatory activity in fish is discussed in this review.
As vertebrates, fishes possess two types of immunity named innate and acquired immunity. The innate parameters play involves significantly in fish immunity and disease resistance. Due to the absence of specialised lymphoid organs, the fish immune system is said to be primitive type and constitutes lymphoid organs and immune cells. It is a non-specific mechanism and functions by complement pathway depending on the target recognition by lectins. Lectins are chemically proteins that can bind to carbohydrates, glycolipids, peptidoglycans, glycoproteins, etc. Major types of fish lectins are galectins, C-type lectins, F-type lectins, rhamnose binding lectins, etc. Major roles played by different lectins include recognition of pathogens, agglutination, opsonisation, complement activation, and phagocytosis. Other than these, molecular functions like splicing of RNA, folding and trafficking of proteins also regulate proliferation of cells. Acquired immunity is the specific immune response acquired during life. It involves humoral immunity, cell-mediated immunity, and immunological memory. Even though the adaptive response in fishes is found to be late, it is considered as important for long-lasting immunity and effective vaccination property. In this chapter, fish immunity is discussed, giving emphasis on lectins and its classes involved during the time of infection.
Over several decades, the disease pattern of intractable disease has changed from acute infection to chronic disease accompanied by immune and metabolic dysfunction. In addition, scientific evidence has shown that humans are holobionts; of the DNA in humans, 1% is derived from the human genome, and 99% is derived from microbial genomes (the microbiome). Extracellular vesicles (EVs) are lipid bilayer-delimited nanoparticles and key messengers in cell-to-cell communication. Many publications indicate that microbial EVs are both positively and negatively involved in the pathogenesis of various intractable diseases, including inflammatory diseases, metabolic disorders, and cancers. Microbial EVs in feces, blood, and urine show significant differences in their profiles between patients with a particular disease and healthy subjects, demonstrating the potential of microbial EVs as biomarkers for disease diagnosis, especially for assessing disease risk. Furthermore, microbial EV therapy offers a variety of advantages over live biotherapeutics and human cell EV (or exosome) therapy for the treatment of intractable diseases. In summary, microbial EVs are a new tool in medicine, and microbial EV technology might provide us with innovative diagnostic and therapeutic solutions in precision medicine.
Recent years have seen unprecedented activity in the development of RNA-silencing oligonucleotide therapeutics for metabolic diseases. Improved oligonucleotide design and optimization of synthetic nucleic acid chemistry, in combination with the development of highly selective and efficient conjugate delivery technology platforms, have established and validated oligonucleotides as a new class of drugs. To date, there are five marketed oligonucleotide therapies, with many more in clinical studies, for both rare and common liver-driven metabolic diseases. Here, we provide an overview of recent developments in the field of oligonucleotide therapeutics in metabolism, review past and current clinical trials, and discuss ongoing challenges and possible future developments. Advances in oligonucleotide design and delivery platforms have enabled the recent approval of several oligonucleotide-based therapies. Here, Goga and Stoffel discuss applications of RNA-silencing oligonucleotide therapeutics in metabolic diseases, recent developments in the field, ongoing challenges and possible future directions.
Many economically important protein-based therapeutics like monoclonal antibodies are glycosylated. Due to the recognized importance of this type of posttranslational modification, glycoengineering of expression systems to obtain highly active and homogenous therapeutics is an emerging field. Although most of the monoclonal antibodies on the market are still produced in mammalian expression platforms, plants are emerging as an alternative cost-effective and scalable production platform that allows precise engineering of glycosylation to produce targeted human glycoforms at large homogeneity. Apart from producing more effective antibodies, pure glycoforms are required in efforts to link biological functions to specific glycan structures. Much is already known about the role of IgG1 glycosylation and this antibody class is the dominant recombinant format that has been expressed in plants. By contrast, little attention has been paid to the glycoengineering of recombinant IgG subtypes and the other four classes of human immunoglobulins (IgA, IgD, IgE, and IgM). Except for IgD, all these antibody classes have been expressed in plants and the glycosylation has been analyzed in a site-specific manner. Here, we summarize the current data on glycosylation of plant-produced monoclonal antibodies and discuss the findings in the light of known functions for these glycans.KeywordsAntibodyGlycanGlycoengineeringGlycoprotein
Nicotiana benthamiana
Plant biotechnologyRecombinant protein
Glycans have been shown to function as versatile molecular signals in cells. This prompted us to look at their roles in endocytosis, endolysosomal system and autophagy. We start by introducing the cell biological aspects of these pathways, the concept of the sugar code, and provide an overview on the role of glycans in the targeting of lysosomal proteins and in lysosomal functions. Moreover, we review evidence on the regulation of endocytosis and autophagy by glycans. Finally, we discuss the emerging concept that cytosolic exposure of luminal glycans, and their detection by endogenous lectins, provides a mechanism for the surveillance of the integrity of the endolysosomal compartments, and serves their eventual repair or disposal.
Significance
The environmental triggers of human colitis are unknown, while current treatments have limited efficacy. To find better therapies, various animal models of colitis have been investigated including studies of inborn genetic defects and chemical toxin ingestion. However, such models do not appear to identify common triggers of disease among human populations. We recently developed a recurrent food-poisoning model of colitis involving the Salmonella bacterial pathogen, a major source of human foodborne illness, and discovered that repeated infections over the adult lifespan cause a progressive intestinal inflammation that leads to a severe and enduring colitis. We have identified the Neu3 neuraminidase as essential in disease onset and provide rationale for the development of Neu3 inhibitors to prevent and treat colitis.
Purpose
To assess the utility of a machine-learning approach for predicting liver function based on technetium-99 m-galactosyl serum albumin (99mTc-GSA) single photon emission computed tomography (SPECT)/CT.
Methods
One hundred twenty-eight patients underwent a 99mTc-GSA SPECT/CT-based liver function evaluation. All were classified into the low liver-damage or high liver-damage group. Four clinical (age, sex, background liver disease and histological type) and 8 quantitative 99mTc-GSA SPECT/CT features (receptor index [LHL15], clearance index [HH15], liver-SUVmax, liver-SUVmean, heart-SUVmax, metabolic volume of liver [MVL], total lesion GSA [TL-GSA, liver-SUVmean × MVL] and SUVmax ratio [liver-SUVmax/heart-SUVmax]) were obtained. To predict high liver damage, a machine learning classification with features selection based on Gini impurity and principal component analysis (PCA) were performed using a support vector machine and a random forest (RF) with a five-fold cross-validation scheme. To overcome imbalanced data, stratified sampling was used. The ability to predict high liver damage was evaluated using a receiver operating characteristic (ROC) curve analysis.
Results
Four indices (LHL15, HH15, heart SUVmax and SUVmax ratio) yielded high areas under the ROC curves (AUCs) for predicting high liver damage (range: 0.89–0.93). In a machine learning classification, the RF with selected features (heart SUVmax, SUVmax ratio, LHL15, HH15, and background liver disease) and PCA model yielded the best performance for predicting high liver damage (AUC = 0.956, sensitivity = 96.3%, specificity = 90.0%, accuracy = 91.4%).
Conclusion
A machine-learning approach based on clinical and quantitative 99mTc-GSA SPECT/CT parameters might be useful for predicting liver function.
Transplantation of islets in Type 1 diabetes is limited by poor islet engraftment into the liver, with 2-3 donor pancreases required per recipient. We aimed to condition the liver to enhance islet engraftment to improve long-term graft function. Diabetic mice received a non-curative islet transplant (n=400 islets) via the hepatic portal vein (HPV) with Fibroblast Growth Factor 7 loaded galactoslyated poly(DL-lactide-co-glycolic acid) (FGF7-GAL-PLGA) particles; 26µm diameter particles specifically targeted the liver, promoting hepatocyte proliferation in short-term experiments: in mice receiving 0.1mg FGF7-GAL-PLGA particles (60ng FGF7) versus vehicle, cell proliferation was induced specifically in the liver with greater efficacy and specificity than subcutaneous FGF7 (1.25mg/kg ×2 doses; ~75µg FGF7). Numbers of engrafted islets and vascularisation were greater in liver sections of mice receiving islets and FGF7-GAL-PLGA particles versus mice receiving islets alone, 72 hours post-transplant. More mice (6 out of 8) that received islets and FGF7-GAL-PLGA particles normalised blood glucose concentrations by 30-days post-transplantation, versus 0 of 8 mice receiving islets alone with no evidence of increased proliferation of cells within the liver at this stage and normal liver function tests. This work shows liver targeted FGF7-GAL-PLGA particles achieve selective FGF7 delivery to the liver promoting islet engraftment to help normalise blood glucose levels with a good safety profile.
A previous paper (Ueno, K., Ohsawa, F., and Natori, S. (1983) J. Biol. Chem. 258, 12210-12214) showed that a storage protein receptor is present in membranes of the pupal fat body of Sarcophaga peregrina, whereas membranes of the larval fat body contain a cryptic receptor, and that 20-hydroxyecdysone is essential for activation of the cryptic receptor to bind and incorporate the storage protein. The present paper reports that the storage protein receptor is a membrane protein of the pupal fat body with a molecular mass of 120 kilodaltons, and that the cryptic receptor present in membranes of the larval fat body is a precursor of the active receptor with a molecular mass of 125 kilodaltons. It is suggested that when 20-hydroxyecdysone is secreted, it activates the process of conversion of the 125-kDa protein to the 120-kDa protein. Concomitant protein synthesis was not apparently necessary for activation of the storage protein receptor, suggesting that the target of 20-hydroxyecdysone in this case is the fat body membrane.
We have studied hepatocytes cultured from Watanabe-heritable hyperlipidemic (WHHL) rabbits, animals that display striking elevation of plasma low density lipoprotein (LDL), spontaneous atherosclerosis, and an absence of LDL receptor activity in cultured fibroblasts. Degradation of LDL by WHHL hepatocytes differed from degradation by normal hepatocytes in several ways: 1) degradation by normal hepatocytes as a function of LDL concentration was curvilinear with a saturable component, while degradation by WHHL hepatocytes was a linear function of concentration; 2) degradation of 125I-labeled LDL by mutant cells was not decreased by excess unlabeled LDL, while degradation by normal cells was; 3) degradation of LDL by normal cells was inhibited by colchicine and chloroquine while degradation by the mutant cells was not; 4) both cell types catabolized LDL at nearly equal rates, but activity of 3-hydroxy-3-methylglutaryl-CoA reductase was suppressed only in the normal cells. These differences are analogous to those previously reported in describing the qualitatively different pathways for receptor-dependent and receptor-independent catabolism of lactosylated and native human LDL in rat hepatocytes. Thus, hepatocytes from WHHL rabbits lack LDL receptor activity. The peripheral and hepatic LDL receptors most likely are products of the same gene or depend for their activity on a single gene product.
Hypersialylation caused by the overexpression of sialyltransferases (STs) is a common feature in cancer that is associated with several characteristics of tumorigenesis. Thus, identifying cancer-associated STs is critical for cancer therapy. However, ST screening has been frequently conducted in cell line models. In this study, we conducted a comprehensive analysis of STs in the clinical database and identified the STs related with the survival of breast cancer patients. RNA sequencing (RNA-Seq) data of 496 patients were obtained from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA). Of the eight mapped STs, ST3GAL5, and ST8SIA1 met the acceptable area under the curve (AUC) criteria for overall survival (OS). Using Kaplan–Meier methods, we determined that high expression of ST8SIA1 was associated with poor 10-year OS in all patients, triple-negative breast cancer (TNBC), and non-TNBC patients, and poor disease-free survival (DFS) rates particularly in TNBC. ST8SIA1 also had superior AUC values in terms of OS/DFS. High ST8SIA1 levels showed a higher risk for poor OS in different groups of patients and a higher risk for poor DFS particularly in TNBC. In summary, we conducted a comprehensive analysis of STs from the clinical database and identified ST8SIA1 as a crucial survival-related ST, which might be a potential therapeutic target for breast cancer and TNBC patients.
RNA interference (RNAi) applications have evolved from experimental tools to study gene function to the development of a novel class of gene-silencing therapeutics. Despite decades of research, it was not until August 2018 that the US FDA approved the first-ever RNAi drug, marking a new era for RNAi therapeutics. Although there are many limitations associated with the inherent structure of RNA, delivery to target cells and tissues remains the most challenging. RNAs are unable to diffuse across cellular membranes due to their large size and polyanionic backbone and, therefore, require a delivery vector. RNAi molecules can be conjugated to a targeting ligand or packaged into a delivery vehicle. Alnylam has used both strategies in their FDA-approved formulations to achieve efficient delivery to the liver. To harness the full potential of RNAi therapeutics, however, we must be able to target additional cells and tissues. One promising target is the folate receptor α, which is overexpressed in a variety of tumors despite having limited expression and distribution in normal tissues. Folate can be conjugated directly to the RNAi molecule or used to functionalize delivery vehicles. In this review, we compare both delivery strategies and discuss the current state of research in the area of folate-mediated delivery of RNAi molecules.
An accurate preoperative evaluation of the hepatic function and application of portal vein embolization in selected patients have helped improve the safety of major hepatectomy. In planning major hepatectomy, however, several issues remain to be addressed. The first is which cut-off values for serum total bilirubin level and prothrombin time should be used to define post-hepatectomy liver failure. Other issues include what minimum future liver remnant (FLR) volume is required; whether the total liver volume measured using computed tomography or the standard liver volume calculated based on the body surface area should be used to assess the adequacy of the FLR volume; whether there is a discrepancy between the FLR volume and function during the recovery period after portal vein embolization or hepatectomy; and how best the function of a specific FLR can be assessed. Various studies concerning these issues have been reported with controversial results. We should also be aware that different strategies and management are required for different types of liver damage, such as cirrhosis in hepatocellular carcinoma, cholangitis in biliary tract cancer, and chemotherapy-induced hepatic injury.
Vutrisiran (ALN-TTRsc02) is a liver-directed, investigational, small interfering ribonucleic acid drug for the treatment of transthyretin (TTR)-mediated amyloidosis. This Phase 1, randomized, single-blind, placebo-controlled, single ascending dose study evaluated the pharmacodynamics, pharmacokinetics, and safety profile of subcutaneously-administered vutrisiran (5-300 mg) in healthy subjects (n = 80). Vutrisiran treatment achieved potent and sustained TTR reduction in a dose-dependent manner, with mean maximum TTR reduction of 57-97%, maintained for ≥90 days post-dose. Vutrisiran was rapidly absorbed (peak plasma concentration 3-5 h post-dose), had a short plasma half-life (4.2-7.5 h), and plasma concentrations increased in a dose-proportional manner. Pharmacodynamic and pharmacokinetic results were similar in Japanese and non-Japanese subjects. Vutrisiran had an acceptable safety profile; the most common treatment-related adverse event was mild, transient injection site reactions in four (6.7%) vutrisiran-treated subjects. The favorable pharmacokinetic, pharmacodynamic, and safety results observed here support vutrisiran's continued clinical development.
FITC-labeled arabinogalactan (FA), pullulan (FP-60), dextran (FD-70) and mannan (FM) were prepared by reaction with FITC in dimethyl sulphoxide according to the method of deBelder and Granath. Hepatic distribution of the FITC-labeled polysaccharides was examined using a specific high-performance size-exclusion chromatography. The microscopic examination also revealed the site-specific distribution of FITC-labeled polysaccharides. Intravenously injected FA was rapidly eliminated from the blood circulation followed by appreciable distribution to the parenchymal liver cells. FP-60 was also endocytosed specifically by the parenchymal liver cells, but FD-70, the same glucan as FP-60, showed a preferential distribution to the sinusoidal lining cells such as Kupffer cells and endothelial cells. Intravenously injected FM was taken up particularly by the sinusoidal lining cells. These results demonstrated that although the polysaccharides were markedly accumulated in the liver, target cell types were quite different from each other depending on the constituent sugar species. It was further suggested that the parenchymal liver cells made the highest relative contribution to the excretion of the polysaccharides not only by means of the receptor-mediated endocytosis, but also by means of non-specific fluid-phase endocytosis.
Givosiran is a small interfering ribonucleic acid agent that was recently approved in the US for the treatment of acute hepatic porphyria (AHP). This Phase 1 study evaluated the safety, pharmacokinetic and pharmacodynamic profile of subcutaneously (SC) administered givosiran in patients with acute intermittent porphyria (AIP), the most common AHP type. Givosiran was rapidly absorbed from the SC injection site with peak plasma concentrations achieved within 0.5 ‐ 5 hours followed by elimination with a short half‐life of 4‐10 hours. Plasma exposures of AS(N‐1)3’ givosiran, an active metabolite with equal potency as givosiran, was 35 ‐ 75%. Givosiran treatment resulted in a rapid and dose‐dependent reduction in urinary aminolevulinic acid (ALA) and porphobilinogen (PBG) towards the upper limit of normal (ULN) in AHP patients. Greater and more sustained reductions in ALA and PBG were achieved with once monthly dosing compared to once quarterly dosing. After monthly dosing, trough ALA levels were reduced to below the ULN, approximately 95% reduction from baseline, at both the 2.5 mg/kg and 5.0 mg/kg doses.
High recurrence and metastasis rates are the major causes of the high mortality of hepatocellular carcinoma (HCC). Circulating tumor cells (CTCs) disseminating into the bloodstream plays an essential role in cancer metastasis. However, since HCC-CTCs are extremely rare, limitations of current detection methods impede accurate discerning HCC-CTCs under complicate biological context. Here, a dual-targeting functionalized rGO film (DTFGF) for specific identifying HCC-CTCs was created via coinstantaneous targeting epithelial cell adhesion molecule (EpCAM) and HCC cell-specific asialoglycoprotein receptor (ASGPR). Anti-EpCAM antibodies and galactose-rhodamine-polyacrylamide nanoparticles (Gal-Rh-PAA NPs) specifically recognizing ASGPR are modified on the surface of a graphene film that quench the rhodamine fluorescence. HCC-CTCs can be captured by anti-EpCAM antibody and endocytose Gal-Rh-PAA NPs, recovering the rhodamine fluorescence. Profiting from accuracy of dual-targeting, less handling steps and high resolution of fluorescence imaging, a simple, rapid and low-cost HCC-CTC enumeration method is established with excellent sensitivity and selectivity than conventional methods. Using DTFGFs, as low as 5 HCC-CTCs were detected in a 1 mL blood sample. Further results revealed that larger HCC-CTCs quantities indicate more advanced stages of HCC in patients. Overall, this work holds great promise for the early diagnosis, prognosis and therapeutic evaluation of HCC.
The asialoglycoprotein receptor (ASGPr) and N-acetylgalactosamine (GalNAc) is one of the most reliable receptor-ligand combinations for delivering antisense oligonucleotides (ASOs) to the liver. Here, we show that a modular GalNAc conjugation strategy allows us to reinforce the activity of the parent, naked 2′,4′-BNA/LNA gapmer targeting apolipoprotein B. The conjugation partly reduced a possible hepatotoxicity of the parent ASO. The structure-activity study revealed the significance of the metabolic susceptibility of the GalNAc moiety to nucleolytic cleavage that results in exposure of the parent gapmer. The broad usefulness of our delivery strategy of ASOs to the liver has been demonstrated.
Ligand‐targeted drug delivery (LTDD) has emerged as an attractive option in the field of oligonucleotide drugs following the great success of N ‐acetylgalactosamine (GalNAc)–conjugated siRNA and antisense oligonucleotides. GalNAc is a well‐known ligand of the asialoglycoprotein receptor (ASGPR), and is classified as a C‐type lectin associated with the metabolism of desialylated glycoproteins. This article describes the synthesis of a non‐nucleosidic monovalent GalNAc phosphoramidite—a useful reagent for facilitating the conjugation of GalNAc epitopes into oligonucleotides using DNA synthesizers—together with some important caveats. The monomeric GalNAc consists of three parts: (1) a GalNAc moiety, (2) a linker moiety, and (3) a trans ‐4‐hydroxyprolinol ( t HP) branch point. The GalNAc moiety and the t HP moiety are coupled via a condensation reaction to prepare the monovalent GalNAc phosphoramidite. © 2019 by John Wiley & Sons, Inc.
Basic Protocol 1 : Synthesis of N ‐acetylgalactosamine ligand
Basic Protocol 2 : Preparation of trans ‐4‐hydroxyprolinol building block
Basic Protocol 3 : Preparation of GalNAc phosphoramidite
Nonalcoholic fatty liver disease (NAFLD) is a significant public health challenge afflicting approximately 1 billion individuals both in the Western world and in the East world. While liver biopsy is considered as gold standard in the diagnosis and staging of liver fibrosis, noninvasive imaging technologies, including ultrasonography, computed tomography, single-photon emission computed tomography (SPECT), magnetic resonance imaging, and positron emission tomography (PET) could offer more sensitive, comprehensive, and quantitative measurement for NAFLD. In this review, we focus on recent development and applications of PET/SPECT molecular probes that enable multispatial/temporal visualization and quantification of physiopathological progress at the molecular level in the NAFLD. We shall also discuss the limitations of current radioligands and future direction for PET/SPECT probe development.
The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = -0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies. SIGNIFICANCE STATEMENT: Although multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.
Glycans in polysaccharides and glycoconjugates of the hydrophilic exterior of all animal cells participate in signal transduction, cellular adhesion, intercellular signaling, and sites for binding of pathogens largely through protein–glycan interactions. Microarrays of defined glycans have been used to study the binding specificities of biologically relevant glycan-binding proteins (GBP), but such arrays are limited by their lack of diversity or relevance to the GBP being investigated. Shotgun glycan microarrays are made up of structurally undefined glycans that were released from natural sources, labeled with bifunctional reagents so that they can be monitored during their purification using multidimensional chromatographic procedures, stored as a tagged glycan library (TGL) and subsequently printed onto microarrays at equal molar concentrations. The shotgun glycan microarray is then interrogated with a biologically relevant GBP and the corresponding glycan ligands can be retrieved from the TGL for detailed structural analysis and further functional analysis. Shotgun glycomics extended the defined glycan microarray to a discovery platform that supports functional glycomic analyses and may provide a useful process for ultimately defining the human glycome.
Sensitive and selective polymeric nanocavities for glycoprotein detection were developed using a newly designed molecular imprinting technique involving post‐imprinting modification (PIM), allowing for the introduction of two different interaction sites and a fluorescent reporter within each nanocavity. α‐Fetoprotein (AFP), a biomarker for hepatocellular carcinoma, was employed as a model glycoprotein. For undertaking PIM‐based molecular imprinting, AFP conjugated with polymerizable groups via disulfide bonding was prepared and then immobilized on a phenylboronic acid‐immobilized substrate by the formation of cyclic diesters between AFP glycans in an oriented immobilization manner, facilitating the creation of homogeneous AFP‐imprinted nanocavities. After surface‐initiated atom transfer radical polymerization with a functional monomer, a comonomer and a crosslinker, the disulfide bonds and cyclic diesters were cleaved to create AFP‐imprinted nanocavities, generating free thiol groups present inside the nanocavities only. A thiol‐reactive fluorescent dye was then added to the nanocavities via in‐cavity PIM, yielding signaling AFP‐imprinted nanocavities capable of transducing binding events into fluorescence changes with the limit of detection of AFP appeared to be 0.27 ng/mL in diluted human serum. These results reveal that the proposed in‐cavity PIM‐based molecular imprinting technique is effective for achieving ELISA‐relevant, antibody‐free sensing systems for glycoproteins.
The human microbiome is known to play an essential role in influencing host health. Extracellular vesicles (EVs) have also been reported to act on a variety of signaling pathways, distally transport cellular components such as proteins, lipids, and nucleic acid, and have immunomodulatory effects. Here we shall review the current understanding of the intersectionality of the human microbiome and EVs in the emerging field of microbiota-derived EVs and their pharmacological potential. Microbes secrete several classes of EVs: outer membrane vesicles (OMVs), membrane vesicles (MVs), and apoptotic bodies. EV biogenesis is unique to each cell and regulated by sophisticated signaling pathways. EVs are primarily composed of lipids, proteins, nucleic acids, and recent evidence suggests they may also carry metabolites. These components interact with host cells and control various cellular processes by transferring their constituents. The pharmacological potential of microbiomederived EVs as vaccine candidates, biomarkers, and a smart drug delivery system is a promising area of future research. Therefore, it is necessary to elucidate in detail the mechanisms of microbiome-derived EV action in host health in a multi-disciplinary manner.
We have reported that hypertrophic adipocytes release extracellular vesicles (EVs) and that number of circulating adipocyte-derived EVs correlated with insulin and homeostasis model assessment-insulin resistance (HOMA-IR) in a pilot study using obese patients. Here, we explored the association between circulating EV level and various metabolic parameters, including obesity and lipid and glucose metabolisms among 203 subjects (76 men, 127 women; median age, 54 years) with or without risk factor for metabolic diseases, who received a 75-g oral glucose tolerance test (OGTT). Circulating EV number was significantly higher in men than in women (p<0.001). Circulating EV number in individuals with impaired OGTT pattern was significantly higher compared to those with normal OGTT pattern (p<0.05). Multiple regression analysis revealed that circulating EV number correlated most strongly and significantly with elevated triglyceride (TG) (t=8.55, p<0.001). Additionally, circulating EV number correlated significantly with homeostasis model assessment-beta cell function (HOMA-β) (t=2.38, p<0.05). Receiver operating characteristic curve revealed the cut off value of EV numbers in individuals with elevated serum TG levels (≧ 150mg/dL) was identified (136,738 EVs/μl of plasma, p<0.001, sensitivity 0.842, false positive rate 0.257). Perilipin and asialoglycoprotein receptor 1 were detected on a part of isolated circulating EVs, indicating EV release from adipocytes and hepatocytes, which were related to lipid and glucose metabolism. Circulating EVs represent a promising metabolic biomarker for lipid and glucose metabolism and have potential for monitoring metabolic status in humans, including individuals without metabolic risk factors.
Chronic hepatitis B virus (HBV) infection poses a significant health challenge due to
associated morbidity and mortality from cirrhosis and hepatocellular cancer that eventually results
in the breakdown of liver functionality. Nanotechnology has the potential to play a pivotal role in
reducing viral load levels and drug-resistant HBV through drug targeting, thus reducing the rate of
evolution of the disease. Apart from tissue targeting, intracellular delivery of a wide range of drugs
is necessary to exert a therapeutic action in the affected organelles. This review encompasses the
strategies and techniques that have been utilized to target the HBV-infected nuclei in liver hepatocytes,
with a significant look at the new insights and most recent advances in drug carriers and their role in
anti-HBV therapy.
Glycosylation is a common co- and post-translational modification that can influence many protein properties including protein folding, stability, immunogenicity, pharmacokinetic behavior, and biological activity. Current expression systems for recombinant glycoproteins typically produce a mixture of different glycans that are often not optimal for a desired function. Therefore, there is a growing need for expression systems allowing the precise control of the glycosylation potential for the production of glycoproteins with defined glycans. Plants offer great advantages for tailoring the glycan profile of recombinant glycoproteins as the glycosylation machinery is less complex compared to mammalian cells and numerous glycan manipulation steps are well-tolerated without affecting biomass formation or recombinant protein production.
The abundant cell surface asialoglycoprotein receptor (ASGPR) is a highly selective receptor found on hepatocytes that potentially can be exploited as a selective shuttle for delivery. Various nucleic acid therapeutics that bind ASGPR are already in clinical development, but this receptor-mediated delivery mechanism can be saturated, which will likely result in reduced selectivity for the liver and therefore increase the likelihood for systemic adverse effects. Therefore, when aiming to utilize this mechanism, it is important to optimize both the administration protocol and the molecular properties. We here present a study using a novel ASGPR-targeted antibody to estimate ASGPR expression, turnover and internalization rates in vivo in mice. Using pharmacokinetic data (intravenous and subcutaneous dosing) and an in-silico target-mediated drug disposition (TMDD) model, we estimate an ASGPR expression level of 1.8 million molecules per hepatocyte. The half-life of the degradation of the receptor was found to be equal to 15 hours and the formed ligand-receptor complex is internalized with a half-life of 5 days. A biodistribution study was performed and confirmed the accuracy of the TMDD model predictions. The kinetics of the ASGPR shows that saturation of the shuttle at therapeutic concentrations is possible; however, simulation allows the dosing schedule to be optimized. The developed TMDD model can be used to support the development of therapies that use the ASGPR as a shuttle into hepatocytes.
RNAi is a powerful tool that can be used to probe gene function as well as for therapeutic intervention. We describe a workflow and methods to identify screen and select potent and specific siRNAs in vitro and in vivo using qPCR-based methods as well as an AAT activity assay. We apply these techniques to a set of siRNAs targeting rat AAT, and use this set to exemplify the cell-based and in vivo data that can be generated using these methods.
Erythropoietin (EPO) is one of the main treatments for anaemic patients, greatly improving their quality of life. In this study, biosimilars Binocrit and a development product, called here CIGB-EPO, were compared to the originator product, Eprex. All three are epoetin alpha products, reputed to have similar glycosylation profiles. The quality, safety and efficacy of this biotherapeutic depend on the following glycosylation critical quality attributes (GCQAs): sialylation, N-glycolyl-neuraminic acid (Neu5Gc) content, branching, N-acetyl-lactosamine (LacNAc) extensions and, arguably, O-acetylation pattern. Reverse-phase ultra high pressure liquid chromatography (RP-UHPLC) analysis of acid-released, 1,2-diamino-4,5-methylenedioxybenzene (DMB) labelled sialic acid derivatives and hydrophilic interaction liquid chromatography (HILIC) in combination with mass spectrometry (HILIC-UHPLC-MS) of procainamide (PROC) labelled N-glycans were the analytical tools used. An automated method for enzymatic release and PROC labelling was applied for the first time to ESA products, which facilitated novel, in-depth characterisation and allowed identification of precise structural features including the location of O-acetyl groups on sialic acid (SA) moieties. Samples were digested by a sialate-O-acetylesterase (NanS) to confirm the presence of O-acetyl groups. It was found that Eprex contained the greatest relative abundance of O-acetylated derivatives, Binocrit expressed the least Neu5Gc, and CIGB-EPO showed the greatest variety of high-mannose-phosphate structures. The sialylation and LacNAc extension patterns of the three ESAs were similar, with a maximum of four N-acetyl-neuraminic acid (Neu5Ac) moieties detected per glycan. Such differences in SA derivatisation, particularly O-acetylation, could have consequences for the quality and safety of a biotherapeutic, as well as its efficacy.
Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by theUnited StatesNational Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.
A surface plasmon resonance sensor whose surface has been modified with boronic acid groups was developed in order to bind and detect transferrin. 4-Mercaptophenylboronic acid (4-MPBA) was utilized to form a self-assembled monolayer on the gold surface of the sensor chip. The surface topography of the modified gold chip was characterized by atomic force microscopy. In solutions of pH 9, the glycosyl groups of transferrin (Trf) quickly and reversibly combine with 4-MPBA on the sensor chip to result in changes of the refractive index. Thus, Trf can be sensed online by monitoring the SPR signal. Under optimized conditions, the sensor has a linear response in the 10 nM to 1.3 μM Trf concentration range, and the limit of detection is 4.4 nM (equivalent to 0.34 μg·mL⁻¹). The results suggest that this 4-MPBA-functionalized sensor is a viable candidate for clinical testing of Trf which is a potential marker for severe diseases such as hypertension, primary renal disease, and diabetes.
Graphical abstractSchematic of a novel SPR sensor modified with boronic acid for the determination of transferrin in human urine with high sensitivity.
A general method is described for the quantitative determination of the binding of glycoproteins, and their enzymatically modified derivatives, to the plasma membranes of rat liver. The assay is based upon the competitive inhibition of the binding phenomenon in an operationally irreversible system. Utilizing this procedure, a quantitative estimate of the ability of the membranes to bind four normally circulating serum glycoproteins, as well as their asialo and agalacto derivatives, has been made. The results obtained extend the previous qualitative observations in vivo and provide the basis for a systematic analysis of the structural determinants involved in the binding process.
The survival time in plasma of incompletely desialylated ceruloplasmin, labeled with ⁶⁴Cu or with tritium, was studied in the rat. Removal of less than 20% of the terminal sialic acid, with consequent exposure of the penultimate galactosyl residues, resulted in the rapid disappearance from the circulation of more than 50% of the injected dose. Statistical analysis of the experimental results by means of the Poisson distribution equation supports the estimate that the exposure of no more than 2 galactosyl residues is sufficient to effect prompt clearance of ceruloplasmin from the circulation. Examination of the specificity of the two neuraminidase preparations used in this study revealed that each of the enzymes possessed a preferential affinity for different sialic acid residues, although the results from each were statistically predictable. Consequently, the removal of any 2 sialic acid residues from 1 molecule of ceruloplasmin appears to be incompatible with the continued survival of that molecule in the circulation.
Analyses of the carbohydrate chains of prothrombin indicated that each chain contained sialic acid, galactose, mannose, and glucosamine in the ratio of 2 to 3:2:3:4 to 6, respectively. Based on the galactose content of whole prothrombin there are four carbohydrate chains per enzyme molecule. Enzymatic degradation of the external carbohydrate indicated that the sequence was
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The inner carbohydrate was resistant to enzymatic attack and consisted of about one-third of the total mannose and one-half of the total N-acetylglucosamine residues. It was shown that the carbohydrate chain was not attached to serine or threonine and that it is probably linked to asparagine. The α and β anomeric forms of galactose were present in about a 1:1 ratio. Glycopeptides containing α- and β-galactosidic bonds could be partially separated, indicating that the two anomers are incorporated into specific carbohydrate chains and are not randomly dispersed. The carbohydrate content and enzymatic degradation revealed few differences between the remainder of the carbohydrate in the α- and β-galactose-containing glycopeptides.
Crystalline ceruloplasmin was substantially freed of sialic acid by the action of neuraminidase. Sequential treatment of asialoceruloplasmin with galactose oxidase and tritiated borohydride resulted in the incorporation of tritium into at least 4 residues of D-galactose of the protein molecule.
A general method is described for the radioactive labeling of proteins containing terminal sialic acid in the carbohydrate moieties. Quantitative conversion of these residues to a radioactive 7-carbon analogue of sialic acid was achieved by sequential periodate oxidation and tritiated borohydride reduction. Evidence is presented to establish the identity of the radioactive derivative as 5-acetamido-3, 5-dideoxy-l-arabino-2-heptulosonic acid and to indicate it as the sole site of tritium incorporation in the carbohydrate chain. Upon injection into rats, labeled preparations of ceruloplasmin and orosomucoid exhibited a normal half-life in the plasma and proved suitable for metabolic studies. The data indicate that the 7-carbon sialic acid analogue mimics sialic acid with respect to hydrolysis by neuraminidase and in regulating survival of glycoproteins in the circulation.
A procedure is described for the radioactive labeling of proteins in vitro in which ³H- or ¹⁴C-methyl groups are attached to the protein amino groups by reductive alkylation. Several proteins and several viruses have been labeled to specific activities of typically 5 x 10⁶ cpm per mg of protein. Some applications are illustrated.
Injection into rabbits of radioactive ceruloplasmin from which sialic acid had been removed enzymatically resulted in a rapid
disappearance of the asialoceruloplasmin from the serum and its appearance in parenchymal cells, but not in Kupffer cells,
of the liver. The rapidity of this transfer was dependent upon the presence of intact galactose residues as the terminal,
nonreducing sugar of the carbohydrate moiety. Intact asialoceruloplasmin, doubly labeled with tritium and 64Cu, was isolated from the liver in good yield 24 min after intravenous injection.
The preparation of a particulate sialyl transferase from rat liver and its properties are described. The enzyme was used,
in the presence of cytidine monophosphate-N-acetylneuraminic acid-1-14C and asialoceruloplasmin, to prepare substantive quantities of radioactive ceruloplasmin. With a completely desialylated
preparation of the protein as acceptor, approximately 70% of the normal complement of N-acetylneuraminic acid was restored, and the reconstituted product was isolated and purified. Upon intravenous injection of
this material into a rabbit, essentially all of the radioactivity disappeared from the circulation and was recoverable from
the liver. With partially desialylated ceruloplasmin as acceptor, a radioactive preparation was obtained wherein the N-acetylneuraminic acid content approximated 85% that of the native protein. Upon injection of this material, 50% of the labeled
ceruloplasmin survived in the serum with a normal half-life of 54 hours. These results are interpreted as indicating that
exposure of only a small number of the total galactosyl residues of ceruloplasmin is sufficient to mark the molecule for removal
from the circulation by the liver.
Glycopeptides were isolated from seven γG myeloma proteins and the γG-immunoglobulins of one normal individual. The composition of each glycopeptide was determined and the structure of the oligosaccharide portion of the molecule established as follows. Sequential enzymatic degradation of the oligosaccharide chains with purified glycosidases established the sequences of the sugars. Serial periodate oxidation and methylation of both intact and partially degraded glycopeptides followed by identification of the methylated sugars by gas chromatography and mass spectrometry established the linkages between sugars.
The carbohydrate composition of the glycopeptides was found to be (in residues per mole of glycopeptide): sialic acid, 0 to 2; fucose, 0.6 to 1.2; galactose, 0.3 to 2.3; N-acetylglucosamine, 3.9 to 6.0; and mannose, 3. The variability in the number of sialic acid, fucose, and galactose residues was shown to be caused by microheterogeneity of the oligosaccharides while the variability in the number of N-acetylglucosamine residues reflects differences in the sequence of the sugars in the "core" of the molecules. Two of the seven myeloma proteins had an additional oligosaccharide chain.
All the glycopeptides are branched oligosaccharides containing two nonreducing termini with the sequence Gal (β1,6)/→ GlcNAc (β1,2)/→ Man. When sialic acid is present, it is linked α2,6 to the galactose residue. Both branches are connected to another mannose residue in the core, which also contains either 2 or 3 N-acetylglucosamine (GlcNAc) residues. The sequence of sugars in the core varied from one glycopeptide to another, being either Man→GlcNAc→ GlcNAc→Asn, Man→[GlcNAc→]GlcNAc→Asn, or Man→ GlcNAc→[GlcNAc→]GlcNAc→Asn. Di-N-acetylchitobiose was isolated from the core of one glycopeptide with a core structure of Man→GlcNAc→GlcNAc→Asn.
These studies establish that homogeneous γG-immunoglobulins of man may have different sequences of sugars in their oligosaccharide chains. The data also show that microheterogeneity of oligosaccharides exists in homogeneous glycoproteins secreted by a single clone of cells.
Evidence is presented to identify plasma membranes of liver as the major locus of binding for circulating glycoproteins. The binding process involves a dual role for sialic acid in that its presence on the membranes is essential, whereas its presence on the glycoprotein is incompatible with binding. Enzymatic restoration of sialic acid residues onto partially desialylated membranes is accompanied by increased binding activity and is suggestive of the presence of an intrinsic membrane-associated sialyltransferase.
In addition to the requirement for sialic acid, the binding of proteins by plasma membranes exhibits an absolute dependence upon the presence of calcium. Changes in the concentration of this ion, within the pH range of 6.0 to 7.8, result in a rapid reversal of the binding process and provide a model system for the study of protein uptake and transport by the intact liver cell.
Sialidase activity towards ganglioside substrate, including hematoside, paralleled activity of 5′-nucleotidase and was found to be enriched in the plasma membrane fractions (Touster, O., Aronson, N. N., Jr., Dulaney, J. T., and Hendrickson, M. (1970) J. Cell. Biol. 47, 604) prepared from rat liver cells. Lesser sialidase activity towards ganglioside substrate was found in fractions (Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J. W., Fowler, S., anddeDuve, C. (1968) J. Cell. Biol. 37, 482) containing the lysosomal marker, acid phosphatase. Concentration of gangliosides, and total bound sialic acid per mg of protein, also paralleled the plasma membrane marker, 5′-nucleotidase. Subcellular fractions were prepared from rat liver cells by a recently developed procedure (Touster et al.) which favors the isolation of the plasma membrane fraction, and by an independent method (Leighton et al.) favoring the isolation of lysosomes. These findings indicate that a major fraction of sialidase with activity towards ganglioside substrate is localized in the plasma membrane of the liver cell, and that ganglioside substrate is also localized in this cellular structure.
When desialylated ceruloplasmin (ASCPN), labeled with ⁶⁴Cu and with ³H in its galactose residues, is injected intravenously into a rat, more than half of the radioactivity can be recovered within minutes in ASCPN precipitated immunochemically from liver homogenates. Both radioautographic studies and measurements of hepatic uptake of ASCPN following blockade of the reticuloendothelial system indicate that ASCPN enters only hepatocytes. Serial measurements on the immunoprecipitated ASCPN indicate that cleavage of ⁶⁴Cu is 80% complete within 30 min of injection, whereas galactose is split from the protein even faster. Sucrose gradient fractionation of liver homogenates from rats treated with Triton WR 1339 or dextran reveals the principal site of the catabolic cleavage of copper and galactose to be lysosomes.
Evidence is presented to indicate a generalized role for the terminal sialic acid residues of circulating glycoproteins. Upon injection into rats, all of the desialylated plasma proteins tested, with the exception of transferrin, were promptly removed from the circulation and were recovered from the liver. The materials examined included orosomucoid, fetuin, ceruloplasmin, haptoglobin, α2-macroglobulin, thyroglobulin, lactoferrin, and the two gonadotropic hormones, human chorionic gonadotropin and follicle-stimulating hormone.
Competitive inhibition of hepatic uptake was demonstrated by the injection of tracer amounts of ⁶⁴Cu-ceruloplasmin together with substantive amounts of the above-mentioned desialylated proteins or the glycopeptides derived from them. Uptake was not inhibited by the fully sialylated protein or their glycopeptides.
Plasma glycoproteins were oxidized with periodic acid and reduced with tritiated KBH4, resulting in macromolecules specifically labeled with tritium on their sialic acid residues. The resulting tritiated seven-carbon analog of N-acetylneuraminic acid, abbreviated [3H]AcNeu, covalently attached as part of the fetuin molecule, [3H-AcNeu]fetuin, had a specific activity of 3.67 counts × min−1× ng−1. It served as a very suitable substrate for the measurement of bacterial and mammalian neuraminidase. Clostridium perfringens neuraminidase had a Km of 9 μM and a V of 50 counts × min−1× h−1× ng−1. [3H-AcNeu]Fetuin was homogeneous on polyacrylamide gel electrophoresis and migrated exactly as native fetuin following treatment by sodium dodecyl-sulfate, mercaptoethanol, and boiling. Mild acid hydrolysis removed 70% of the radioactivity, which chromatographed as a homogeneous product utilizing butylacetate, acetic acid and water (3:2:1, v/v/v) as the solvent system.
Mammalian neuraminidase was located in a particulate fraction of rat liver. The enzyme had an acid pH optimum and may be lysosomal in origin. The use of [3H-AcNeu7]glycoprotein substrates increased the sensitivity of measurement of the released sialic acid and provided a more reliable estimate of neuraminidase levels in mammalian tissue sources.
Livers from rats fed a vitamin-K-deficient diet were also assayed for activity. The results indicated that vitamin-K-deficient animals had a slightly elevated amount of neuraminidase activity with [3H-AcNeu7]prothrombin substrate. Prothrombin, unlabeled or labeled, was not extensively degraded by Cl. perfringens neuraminidase. Therefore, either differences exist between bacterial enzyme and mammalian enzyme in the substrate specificities, or the mammalian enzyme source may contain more than one neuraminidase, i.e. one capable of the degradation of the sialic acid residues of fetuin and another capable of the degradation of the sialic acid residues of prothombin.
The survival time in plasma of incompletely desialylated ceruloplasmin, labeled with 64Cu or with tritium, was studied in the rat. Removal of less than 20% of the terminal sialic acid, with consequent exposure
of the penultimate galactosyl residues, resulted in the rapid disappearance from the circulation of more than 50% of the injected
dose. Statistical analysis of the experimental results by means of the Poisson distribution equation supports the estimate
that the exposure of no more than 2 galactosyl residues is sufficient to effect prompt clearance of ceruloplasmin from the
circulation. Examination of the specificity of the two neuraminidase preparations used in this study revealed that each of
the enzymes possessed a preferential affinity for different sialic acid residues, although the results from each were statistically
predictable. Consequently, the removal of any 2 sialic acid residues from 1 molecule of ceruloplasmin appears to be incompatible
with the continued survival of that molecule in the circulation.
The effects of progressive removal of sialic acid (desialylation) on the immunological and biological activity of HCG, as measured by radioimmunoassay (RIA), ovarian ascorbic acid depletion assay (OAAD) and ventral prostate weight assay (VPW), have been evaluated. Two highly purified HCG preparations with potencies of about 12,000 IU/mg (bioassay, 2nd Int. Std. HCG) and about 5000 IU/mg (immunoassay, 2nd Int. Std. HCG), were used in these studies. Progressive desialylation was achieved by treating the HCG with varying amounts of Clostridium perfringens neuraminidase. Desialylation did not affect immunological activity. Progressive desialylation up to 25 % reduced biological activity by approximately 75% as measured by OAAD and VPW. Desialylation from 25 to 62% reduced biological activity further, the reduction being much more pronounced when measured by VPW than by OAAD, resulting in a considerable increase in the index of discrimination OAAD /VPW. Desialylation from 62 to 100% did not affect biological activity further, giving preparations with a low but significant biological potency with an index of discrimination OAAD /VPW of around 50. (Endocrinology 88: 456, 1971)
A new technique for labeling human chorionic gmadotropin (HCG) is described. Tritium was introduced into the molecule by reduction with tritiated borohydride after oxidation with periodate. Immunologic and biologic activity was preserved. MATERIALS AND METHODS Labeling. Oxidation of 19.6 mg of HCG in 4 ml of 0.1 M Na acetate, pH 5.6, was carried out with 50 μmoles potassium periodate at room temperature. After 30 minutes, 100 umoles glucose was added to reduce excess periodate. After dialyzing overnight in 0.1 M K2HPO4, pH 7.5, at 3 C, one-half of the preparation was reduced with 9.25 umoles of tritiated potassium borohydride (324 μCi/μmole) dissolved in 0.2 ml of 0.01 M KOH. To assure reduction of all oxidized groups, an additional 37 umoles of unlabeled borohydride was added. After adjusting the pH (5.0 to 5.5) with acetic acid, the preparation was dialyzed in cold distilled water overnight and lyophilized. All preparations were assayed by either the mouse uterine weight (MUW) or ventral prostate weight...
A general method is described for the radioactive labeling of proteins containing terminal sialic acid in the carbohydrate
moieties. Quantitative conversion of these residues to a radioactive 7-carbon analogue of sialic acid was achieved by sequential
periodate oxidation and tritiated borohydride reduction. Evidence is presented to establish the identity of the radioactive
derivative as 5-acetamido-3, 5-dideoxy-l-arabino-2-heptulosonic acid and to indicate it as the sole site of tritium incorporation in the carbohydrate chain. Upon
injection into rats, labeled preparations of ceruloplasmin and orosomucoid exhibited a normal half-life in the plasma and
proved suitable for metabolic studies. The data indicate that the 7-carbon sialic acid analogue mimics sialic acid with respect
to hydrolysis by neuraminidase and in regulating survival of glycoproteins in the circulation.
Follicle-stimulating hormone was labeled with tritiated borohydride after initially oxidizing the hormone with periodate. The radioactive label was introduced into the altered sialic acid fragment, NAN-7, which remained susceptible to Clostridium perfringens neuraminidase. Fifty per cent of biologic and immunologic activities of native FSH was preserved. When 3H-FSH was injected into the tail vein of pseudopregnant or intact immature female Sprague Dawley rats, maximum ovarian concentration of 3H occurred within 5 min in the pseudopregnant and 1 hr in the immature rat but did not exceed plasma 3H concentration until 3–4 hr after injection. The tritiated FSH preparation remained biologically active for more than 4 months after labeling. The long half-life of tritium is an added advantage. (Endocrinology 89: 1356, 1971)
A specific radioactive label was introduced into the sialoglycoproteins of the erythrocyte membrane by sequential sodium periodate oxidation and tritiated sodium borohydride reduction. This was achieved whether the sialoglycoproteins were isolated, present within the intact erythrocyte, or the isolated erythrocyte membranes. The label is found in the oligosaccharide chains of the sialoglycoproteins predominantly in residues which were formerly those of bound sialic acid. The method appears selective for the sialoglycoproteins and certain, as yet unidentified lipid components.
The specific binding of labeled gonadotropin to subcellular fractions of the rat testis has been utilized in the development of a sensitive radioligand assay system for LH and HCG. The cell-free supernatant of the fragmented interstitial cell fraction was found to contain high affinity gonadotropin receptors, which showed rapid and specifically reversible binding of ¹²⁵I-labeled LH and HCG. For binding assays, satisfactory results were also obtained with the fraction prepared by centrifugation at 1500 g after homogenization of the whole testis. Assays were performed by incubation of tracer hormone with standards or samples and binding fraction for 2 hr at 37 C, or 16 hr at 24 C. Separation of bound and free tracer was performed by centrifugation and washing of the particulate receptor fraction, followed by measurement of the bound radioactivity. Parallel dose-response curves were obtained with human pituitary LH (LER 907), human urinary LH, and human chorionic gonadotropin, while ovine, bovine, and rat LH gave parallel standard curves of slightly lower slope than that of the human hormones. Satisfactory standard curves for LH and HCG were obtained over the range 10-500 mU. The presence of LH-HCG receptors with similar characteristics has also been demonstrated in the rat ovary. Radioligand-receptor gonadotropin assays based on binding to gonadal cell fractions are extremely rapid and sensitive, combining the convenience and precision of binding assays with the biological specificity characteristic of the receptor site.
Analyses of the carbohydrate chains of prothrombin indicated that each chain contained sialic acid, galactose, mannose, and
glucosamine in the ratio of 2 to 3:2:3:4 to 6, respectively. Based on the galactose content of whole prothrombin there are
four carbohydrate chains per enzyme molecule. Enzymatic degradation of the external carbohydrate indicated that the sequence
was
[see PDF for equation]
The inner carbohydrate was resistant to enzymatic attack and consisted of about one-third of the total mannose and one-half
of the total N-acetylglucosamine residues. It was shown that the carbohydrate chain was not attached to serine or threonine and that it
is probably linked to asparagine. The α and β anomeric forms of galactose were present in about a 1:1 ratio. Glycopeptides
containing α- and β-galactosidic bonds could be partially separated, indicating that the two anomers are incorporated into
specific carbohydrate chains and are not randomly dispersed. The carbohydrate content and enzymatic degradation revealed few
differences between the remainder of the carbohydrate in the α- and β-galactose-containing glycopeptides.
A lyophilized preparation of a particulate fraction of rabbit liver homogenate exhibits specific binding affinity for a desialylated glycoprotein comparable to that found in rat liver plasma membranes. Treatment of this preparation with Triton X-100 solubilizes the receptor sites which are capable of retaining their specific binding activity in solution.
The effects of removal of the terminal sialic acid and galactose residues of hCG on the immunological and biological properties of the hormone were studied in order to further clarify the role of the carbohydrate moieties in the action of glycoprotein hormones on their target tissues. hCG treated sequentially with neuraminidase and β–galactosidase was evaluated for biological in vivoactivity, for binding activity by radioimmunoassay and radioligand—receptor assay, and for in vitro biological activity by assay of testosterone production by the isolated rat testis. Metabolism of the asialo— and asialo—agalacto—hCG preparations labeled with 125I was compared with that of the intact hormone after intravenous injection. The asialo—agalacto—hCG preparation possessed biological activity in vivo of 2.3 IU/mg, comparable to that of the desialylated hormone (0.7 IU/mg) and considerably below that of the stored intact hormone (7400 IU/mg). By contrast, the ability of the modified hormones to compete with intact hCG ...
The glycopeptides of fetuin and γ G immunoglobulin have been covalently linked to lysozyme and albumin with the coupling reagent toluene 2,4 diisocyanate. When the fetuin glycopeptide-protein compounds were treated with neuraminidase to remove sialic acid, and then injected into rats, the coupled compounds were rapidly and selectively removed from the circulation by the liver, most likely by the hepatocytes. Removal of the exposed galactose residues of the asialofetuin-proteins with β-galactosidase prevented this clearance. Neither the native proteins nor the γ G glycopeptide-proteins are taken up by the liver, demonstrating the specificity of the phenomenon. Once the asialofetuin-protein complexes enter the hepatocytes, they are degraded within two hours.
Native bovine prothrombin was shown to have a circulatory half-life of 90 minutes when injected into the circulatory system of rats. Removal of sialic acid resulted in a circulatory half-life of only 9 minutes and further enzymatic degradation of the carbohydrate restored the circulatory half-life to more nearly that of intact prothrombin. These observations broaden the generality of this phenomenon displayed by some other glycoproteins and therefore the possibility that sialic acid may function in determining the circulatory life of glycoproteins.
The marked reduction of biological activity after desialylation of human luteinizing hormone and chorionic gonadotropin is not accompanied by a corresponding loss of activity in vitro. The desialylated gonadotropins show increased affinity for gonadal binding sites, and retain a considerable degree of steroidogenic activity when incubated with the rat testis in vitro. These findings indicate that the sialic acid residues of gonadotropic hormones are not essential for biological activity at the target cell level.
A specific radioimmunoassay was used to measure concentrations of immunoreactive hormone in plasma samples taken at intervals following injection of intact and variably desialylated preparations of highly purified human chorionic gonadotropin (HCG) into pseudopregnant rats. Desialylation of HCG up to 25% decreased plasma half-life by 50% (from 25 to 11 min). Removal of sialic acid over the range of 25–62% resulted in a more profound reduction in plasma t½ (from 11 to less than 1 min) but desialylation greater than 62 % was without additional effect. The observations suggest that previously observed decrements in biologic activity following progressive desialylation of HCG result, in part, from changes in plasma half-life of the hormone. (Endocrinology 89: 11, 1971)
Precipitating, complement-fixing and neutralizing antibody was produced in rabbits by immunization with a partially purified human pituitary FSH preparation. Apparent serological specificity of this antiserum was increased after absorption with human serum and HCG. Attempts were made to examine immunologic carbohydrate determinant groups of human pituitary FSH by complement-fixation inhibition reactions between the absorbed antiserum and homogeneous human pituitary FSH preparations, using mono- and disaccharides, and by treatment of the hormone preparations with several carbohydrate-hydrolyzing enzymes. Of the saccharides tested, galactose, β-methyl- D-galactopyranoside lactose and melibiose were active in the inhibition test, and the disaccharides were more effective than the monosaccharides on an equimolar basis. Ovarian weight augmentation activity of FSH was completely abolished, but its complement-fixing property was not affected after treatment with neuraminidase. Both biologic and immunologic properties of FSH were resistant to either lysozyme or β-glucosidase. The complement-fixing property was hardly affected by combined treatment of FSH with neuraminidase and lysozyme but was reduced with increasing time of incubation with neuraminidase plus β-glucosidase. From these results it is tentatively concluded that neuraminic acid is essential for biologic activity but is not involved in the antigenic structure of human pituitary FSH and that a D-galactopyranosyl unit is a constituent of the antigenic determinant groups. (Endocrinology 85:330,1969)
Precipitating, complement-fixing and neutralizing antibody was produced in rabbits by immunization with a partially purified human pituitary FSH preparation. Apparent serological specificity of this antiserum was increased after absorption with human serum and HCG. Attempts were made to examine immunologic carbohydrate determinant groups of human pituitary FSH by complement-fixation inhibition reactions between the absorbed antiserum and homogeneous human pituitary FSH preparations, using mono- and disaccharides, and by treatment of the hormone preparations with several carbohydrate-hydrolyzing enzymes. Of the saccharides tested, galactose, β-methyl- D-galactopyranoside lactose and melibiose were active in the inhibition test, and the disaccharides were more effective than the monosaccharides on an equimolar basis. Ovarian weight augmentation activity of FSH was completely abolished, but its complement-fixing property was not affected after treatment with neuraminidase. Both biologic and immunologic properties of FSH were resistant to either lysozyme or β-glucosidase. The complement-fixing property was hardly affected by combined treatment of FSH with neuraminidase and lysozyme but was reduced with increasing time of incubation with neuraminidase plus β-glucosidase. From these results it is tentatively concluded that neuraminic acid is essential for biologic activity but is not involved in the antigenic structure of human pituitary FSH and that a D-galactopyranosyl unit is a constituent of the antigenic determinant groups. (Endocrinology 85:330,1969)
Crystalline ceruloplasmin was substantially freed of sialic acid by the action of neuraminidase. Sequential treatment of asialoceruloplasmin
with galactose oxidase and tritiated borohydride resulted in the incorporation of tritium into at least 4 residues of D-galactose of the protein molecule.