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Proteoglycans present in proteoglycan-enriched urine detected by peptide LC-MS 2 from an in-solution tryptic digest and ranked by molecular mass search score (MOWSE score)
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Inter-α-trypsin inhibitor (IαI) is a complex comprising two heavy chains (HCs) that are covalently bound by an ester bond to chondroitin sulfate (CS), which itself is attached to Ser-10 of bikunin. IαI is essential for the trans-esterification of HCs onto hyaluronan (HA). This process is important for the stabilization of HA-rich matrices during ov...
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... 2 contained 10% Di-0S, 30% Di-4S, and 20% Di-6S disaccharides and 5% 4S-GalNAc and 6S-GalNAc with the remainder (35%) being disulfated disac- charides that were a mixture of Di-2,4S, Di-2,6S, and Di- 4,6S. Although the fractions were not purified for bikunin before disaccharide compositional analysis, peptide identifica- tion of proteoglycan-enriched urine by mass spectrometry indi- cated that bikunin was the most abundant CS proteoglycan present in this sample (Table 1). ...
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Background:
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Citations
... The transfer of HCs from the CS chain of bikunin to the C-6 of the GlcNAc residues of cell-secreted HA, by a transesterification reaction to form the SHAP-HA complex, requires the formation of a covalent intermediate between HC and the transferase tumor necrosis factor stimulated gene-6 protein (TSG-6), followed by the release of bikunin [23][24][25]. In this process, the bikunin CS chain sulfation pattern has been reported to be crucial for the transesterification of HCs to HA [26]. ...
Bikunin is a small chondroitin sulfate proteoglycan (PG) with Ser-protease inhibitory activity that plays pleiotropic roles in health and disease. It is involved in several physiological processes including stabilization of the extracellular matrix (ECM) of connective tissues and key reproductive events. Bikunin is also implicated in both acute and chronic inflammatory conditions and represents a non-invasive circulating and/or urinary (as Urinary Trypsin Inhibitor or UTI) biomarker. It exerts inhibitory effects on urokinase-type plasminogen activator (uPA) and its receptor (uPAR) mediating tumor invasiveness by a down-regulation of uPA mRNA expression, thus representing an anti-metastatic agent. However, only limited data on its potential as a diagnostic and/or prognostic marker of cancer have been reported so far. Recent technological advances in mass spectrometry-based proteomics have provided researchers with a huge amount of information allowing for large-scale surveys of the cancer proteome. To address such issues, we analyzed bikunin expression data across several types of tumors, by using UALCAN proteogenomic analysis portal. In this article we critically review the roles of bikunin in human pathobiology, with a special focus on its inhibitory effects and mechanisms in cancer aggressiveness as well as its significance as cancer circulating biomarker.
... This suggests that the recurrence scores track a free GAGome feature that may causally be implicated with RCC. However, other factors may cause the here-observed aberrations in plasma and urine CS that may be specific or less to RCC, like inflammation-associated proteoglycans [26], glycocalyx degradation [27,28], or renal and hepatic catabolism [29]. It will be important to elucidate the role of these factors to both improve the performance of the scores and understand potential sources of false positives and negatives. ...
Background:
No liquid biomarkers are approved in renal cell carcinoma (RCC), making early detection of recurrence in surgically treated nonmetastatic (M0) patients dependent on radiological imaging. Urine- and plasma free glycosaminoglycan profiles-or free GAGomes-are promising biomarkers reflective of RCC metabolism.
Objective:
To explore whether free GAGomes could detect M0 RCC recurrence noninvasively.
Design setting and participants:
Between June 2016 and February 2021, we enrolled a prospective consecutive series of patients elected for (1) partial or radical nephrectomy for clinical M0 RCC (cohort 1) or (2) first-line therapy following RCC metachronous metastatic recurrence (cohort 2) at Sahlgrenska University Hospital, Gothenburg, Sweden. The study population included M0 RCC patients with recurrent disease (RD) versus no evidence of disease (NED) in at least one follow-up visit. Plasma and urine free GAGomes-consisting of 40 chondroitin sulfate (CS), heparan sulfate, and hyaluronic acid (HA) features-were measured in a blinded central laboratory preoperatively and at each postoperative follow-up visit until recurrence or end of follow-up in cohort 1, or before treatment start in cohort 2.
Outcome measurements and statistical analysis:
We used Bayesian logistic regression to correlate GAGome features with RD versus NED and with various histopathological variables. We developed three recurrence scores (plasma, urine, and combined) proportional to the predicted probability of RD. We internally validated the area under the curve (AUC) using bootstrap resampling. We performed a decision curve analysis to select a cutoff and report the corresponding net benefit, sensitivity, and specificity of each score. We used univariable analyses to correlate each preoperative score with recurrence-free survival (RFS).
Results and limitations:
Of 127 enrolled patients in total, 62 M0 RCC patients were in the study population (median age: 63 year, 35% female, and 82% clear cell). The median follow-up time was 3 months, totaling 72 postoperative visits -17 RD and 55 NED cases. RD was compatible with alterations in 14 (52%) of the detectable GAGome features, mostly free CS. Eleven (79%) of these correlated with at least one histopathological variable. We developed a plasma, a urine, and a combined free CS RCC recurrence score to diagnose RD versus NED with AUCs 0.91, 0.93, and 0.94, respectively. At a cutoff equivalent to ≥30% predicted probability of RD, the sensitivity and specificity were, respectively, 69% and 84% in plasma, 81% and 80% in urine, and 80% and 82% when combined, and the net benefit was equivalent to finding an extra ten, 13, and 12 cases of RD per hundred patients without any unnecessary imaging for plasma, urine, and combined, respectively. The combined score was prognostic of RFS in univariable analysis (hazard ratio = 1.90, p = 0.02). Limitations include a lack of external validation.
Conclusions:
Free CS scores detected postsurgical recurrence noninvasively in M0 RCC with substantial net benefit. External validity is required before wider clinical implementation.
Patient summary:
In this study, we examined a new noninvasive blood and urine test to detect whether renal cell carcinoma recurred after surgery.
... At first, bikunin (also known as protein AMBP) was used as a model CSPG since it was relatively well characterized, available in large quantities from human urine and also used in some countries as a pharmaceutical agent to treat acute pancreatitis (Ly et al., 2011;Lord et al., 2020). Human bikunin has a single CS chain of 27-39 monosaccharides attached to the N-terminal end (Ser-10) of the core protein (Lord et al., 2013). We incubated pharmaceutical grade bikunin with chondroitinase ABC that generated free disaccharides and a residual hexasaccharide structure still attached to the core protein. ...
Chondroitin sulfate proteoglycans (CSPGs) are found at cell surfaces and in connective tissues, where they interact with a multitude of proteins involved in various pathophysiological processes. From a methodological perspective, the identification of CSPGs is challenging, as the identification requires the combined sequencing of specific core proteins, together with the characterization of the CS polysaccharide modification(s). According to the current notion of CSPGs, they are often considered in relation to a functional role in which a given proteoglycan regulates a specific function in cellular physiology. Recent advances in glycoproteomic methods have, however, enabled the identification of numerous novel chondroitin sulfate core proteins, and their glycosaminoglycan attachment sites, in humans and in various animal models. In addition, these methods have revealed unexpected structural complexity even in the linkage regions. These findings indicate that the number and structural complexity of CSPGs are much greater than previously perceived. In light of these findings, the prospect of finding additional CSPGs, using improved methods for structural and functional characterizations, and studying novel sample matrices in humans and in animal models is discussed. Further, as many of the novel CSPGs are found in low abundance and with not yet assigned functions, these findings may challenge the traditional notion of defining proteoglycans. Therefore, the concept of proteoglycans is considered, discussing whether “a proteoglycan” should be defined mainly on the basis of an assigned function or on the structural evidence of its existence.
... Bikunin is expressed in brain tissue (Takano et al., 1999;Kim et al., 2020) and accumulates in brain tumors. Bikunin CS chains contain embedded disulfated CS-D motifs (Lord et al., 2013(Lord et al., , 2020. A related Kunitz protease inhibitor, placental bikunin (hepatocyte growth factor activator inhibitor type-2) has been reported to inhibit glioblastoma tumor invasion (Hamasuna et al., 2001), however, this is a dissimilar protein to serum bikunin. ...
Chondroitin sulfate (CS) is the most abundant and widely distributed glycosaminoglycan (GAG) in the human body. As a component of proteoglycans (PGs) it has numerous roles in matrix stabilization and cellular regulation. This chapter highlights the roles of CS and CS-PGs in the central and peripheral nervous systems (CNS/PNS). CS has specific cell regulatory roles that control tissue function and homeostasis. The CNS/PNS contains a diverse range of CS-PGs which direct the development of embryonic neural axonal networks, and the responses of neural cell populations in mature tissues to traumatic injury. Following brain trauma and spinal cord injury, a stabilizing CS-PG-rich scar tissue is laid down at the defect site to protect neural tissues, which are amongst the softest tissues of the human body. Unfortunately, the CS concentrated in gliotic scars also inhibits neural outgrowth and functional recovery. CS has well known inhibitory properties over neural behavior, and animal models of CNS/PNS injury have demonstrated that selective degradation of CS using chondroitinase improves neuronal functional recovery. CS-PGs are present diffusely in the CNS but also form denser regions of extracellular matrix termed perineuronal nets which surround neurons. Hyaluronan is immobilized in hyalectan CS-PG aggregates in these perineural structures, which provide neural protection, synapse, and neural plasticity, and have roles in memory and cognitive learning. Despite the generally inhibitory cues delivered by CS-A and CS-C, some CS-PGs containing highly charged CS disaccharides (CS-D, CS-E) or dermatan sulfate (DS) disaccharides that promote neural outgrowth and functional recovery. CS/DS thus has varied cell regulatory properties and structural ECM supportive roles in the CNS/PNS depending on the glycoform present and its location in tissue niches and specific cellular contexts. Studies on the fruit fly, Drosophila melanogaster and the nematode Caenorhabditis elegans have provided insightful information on neural interconnectivity and the role of the ECM and its PGs in neural development and in tissue morphogenesis in a whole organism environment.
... In addition, another study has reported that GATA6 enhanced the progression of PC by activating the Wnt signaling pathway via regulating DKK1 [23]. Inter-α-trypsin inhibitors (ITIs) are a family of serine protease inhibitors which have one light chain and various heavy chains [24,25]. ITI heavy chain 5 (ITIH5) was revealed to have close correlation with tumor suppression in diverse kinds of cancers [26][27][28]. ...
Long noncoding RNAs (lncRNAs) are implicated tumor development in a range of different cancers, including pancreatic cancer (PC). Cancer stem cells (CSCs), a drug-resistant cancer cell subset, drive tumor progression in PC. In this work, we aimed to investigate the mechanism by which lncRNA LINC00261 affects the biological functions of CSCs during the progression of PC. Microarray analysis of differentially expressed genes and lncRNAs suggested that LINC00261 is downregulated in PC. Both LINC00261 and ITIH5 were confirmed to be downregulated in PC cells and PC stem cells. Gain-of-function and loss-of-function investigations were performed to analyze their effects on cell proliferation, drug resistance, cell cycle distribution, self-renewal, invasion, and ultimately overall tumorigenicity. These experiments revealed that the expression of stem cell markers was reduced, and cell proliferation, self-renewal ability, cell invasion, drug resistance, and tumorigenicity were all suppressed by upregulation of LINC00261 or ITIH5. The results of dual-luciferase reporter gene, ChIP, and RIP assays indicated that LINC00261 binds directly to GATA6, increasing its activity at the ITIH5 promoter. The presence of LINC00261 and GATA6 inhibited the self-renewal and tumorigenesis of PC stem cells, while silence of ITIH5 rescued those functions. Collectively, this study identifies the tumor suppressive activity of LINC00261 in PC, showing that this lncRNA limits the functions of PC stem through an ITIH5/GATA6 regulatory pathway.
... Proteoglycan-enriched fractions in 25 mM ammonium bicarbonate were reduced (10 mM DTT, 10 min, 95 C), alkylated (25 mM iodoacetamide, 20 min, room temperature), and digested (sequencing grade trypsin, 20 μg/ml, 16 h, 37 C). Samples were subjected to peptide analysis by liquid chromatography-tandem mass spectrometry (LC-MS 2 ) as previously described (74). Samples were analyzed by LC-MS 2 using an LTQ mass spectrometer (Thermo Fisher Scientific). ...
The retention of low-density lipoprotein (LDL) is a key process in the pathogenesis of atherosclerosis, and largely mediated via smooth muscle cell-derived extracellular proteoglycans including the glycosaminoglycan chains. Macrophages can also internalize lipids via complexes with proteoglycans. However, the role of polarized macrophage-derived proteoglycans in binding LDL is unknown and important to advance our understanding of the pathogenesis of atherosclerosis. We therefore examined the identity of proteoglycans, including the pendent glycosaminoglycans, produced by polarized macrophages to gain insight into the molecular basis for LDL binding. Using the quartz crystal microbalance with dissipation monitoring technique, we established that classically activated macrophage (M1)- and alternatively activated macrophage (M2)-derived proteoglycans bind LDL via both the protein core and heparan sulfate (HS) in vitro. Among the proteoglycans secreted by macrophages, we found perlecan was the major protein core that bound LDL. In addition, we identified perlecan in the necrotic core as well as the fibrous cap of advanced human atherosclerotic lesions in the same regions as HS and colocalized with M2 macrophages, suggesting a functional role in lipid retention in vivo. These findings suggest that macrophages may contribute to LDL retention in the plaque by the production of proteoglycans; however, their contribution likely depends on both their phenotype within the plaque and the presence of enzymes, such as heparanase, that alter the secreted protein structure.
... 39,59 More recently, with the advent of different purification protocols and sources of bikunin, it was discovered that the CS chain is variable in both chain length and degree of sulfation and can contain unsulfated, monosulfated, and disulfated disaccharides. 39,[60][61][62] In addition, the linkage region can be variably sulfated. 59,[62][63][64][65][66][67][68][69] ...
... The CS chain attached to bikunin is essential for the transesterification of its pendant HCs to HA in the formation of HA·HC. 46,80 In addition, the structure of the CS chain modulates the extent of HA·HC formation, with more highly sulfated CS chains supporting a greater extent of HA·HC formation than lower sulfated CS, 61 suggesting a mechanism for control of this process. ...
Inter-α-trypsin inhibitor (IαI) family members are ancient and unique molecules that have evolved over several hundred million years of vertebrate evolution. IαI is a complex containing the proteoglycan bikunin to which heavy chain proteins are covalently attached to the chondroitin sulfate chain. Besides its matrix protective activity through protease inhibitory action, IαI family members interact with extracellular matrix molecules and most notably hyaluronan, inhibit complement, and provide cell regulatory functions. Recent evidence for the diverse roles of the IαI family in both biology and pathology is reviewed and gives insight into their pivotal roles in tissue homeostasis. In addition, the clinical uses of these molecules are explored, such as in the treatment of inflammatory conditions including sepsis and Kawasaki disease, which has recently been associated with severe acute respiratory syndrome coronavirus 2 infection in children:
... 39 To better understand the structural requirement of bikunin on its inflammatory responses, bikunin was treated with chondroitinase ABC to cleave off the endogenous CS chain producing the core protein lacking the CS chain. 40 In addition, bikunin was treated with actinase E to completely digest the core protein backbone generating the bikunin associated CS chain, which mainly consists of CS-A 9 as confirmed by our NMR and MS analysis. The abilities of macrophages to respond to LPS upon incubation with bikunin, core protein, synthetic CS 24-mer 1, commercially available CS-A polysaccharide, and bikunin CS were determined by quantifying the amounts of TNF-α released from macrophages using an enzyme linked immunosorbent assay (ELISA) (Figure 3). ...
Bikunin, a chondroitin sulfate (CS) proteoglycan clinically used to treat acute inflammation and sepsis, contains a CS chain with more than 20 monosaccharide units. To understand the function of the CS chain of bikunin, synthesis of long CS chains is needed. After exploring multiple glycosylation approaches and protective group chemistry, we report herein the successful generation of the longest CS chain to date (24-mer) in an excellent overall yield on a multi-mg scale. The anti-inflammatory activities of both bikunin and the synthetic 24-mer were determined, and the results demonstrate that both the glycan and the core protein are important for anti-inflammatory activities of bikunin by reducing macrophage production of proinflammatory cytokines.
... The BAP solution comprised bovine serum albumin (BSA), human apolipoprotein (ApoA1, Athens), human complement protein C3 (C3, CompTech), IgG (Sigma), transferrin (T, Sigma), and inter-alpha trypsin inhibitor (IaI) [13], [16]. The IaI was isolated from human plasma following previously described procedures [29]. AP consisted of BSA supplemented with amino acids according to previously published methods [15]. ...
... Alternatively, it is suggested that insufficient dilution of salt residues from IaI isolation, may contribute to the drop in impedance. Isolation of IaI requires high concentrations of NaCl (1M) to retrieve the protein from the column [29]. However, further testing with different batches, where serial dilution washes during protein isolation were triplicated, resulted in the same drop in impedance effect (results not shown). ...
Objective:
This study evaluated subthreshold biphasic stimulation pulses as a strategy to stabilize electrode impedance via control of protein adsorption. Following implantation, cochlear electrodes undergo impedance fluctuations thought to be caused by protein adsorption and/or inflammatory responses. Impedance increases can impact device power consumption, safe charge injection limits, and long-term stability of electrodes.
Methods:
Protein-mediated changes in polarization impedance (Zp) were measured by voltage transient responses to biphasic current pulses and electrochemical impedance spectroscopy, with and without protein solutions. Four subthreshold stimulation regimes were studied to assess their effects on protein adsorption and impedance; (1) symmetric charge-balanced pulses delivered continuously, (2) at 10% duty cycle, (3) at 1% duty cycle, and (4) an asymmetric charge balanced pulse delivered continuously with a cathodic phase twice as long as the anodic phase.
Results:
The Zp of electrodes incubated in protein solutions without stimulation for 2 hours increased by between ~28% and ~55%. Subthreshold stimulation reduced the rate at which impedance increased following exposure to all protein solutions. Decreases in Zp were dependent on the type of protein solution and the stimulation regime. Subthreshold stimulation pulses were more effective when delivered continuously compared to 1% and 10% duty cycles.
Conclusion:
These results support the potential of subthreshold stimulation pulses to mitigate protein-mediated increase in impedance.
Significance:
This research highlights the potential of clinically translatable stimulation pulses to mitigate perilymph protein adsorption on cochlear electrodes, a key phenomenon precursor of the inflammatory response.
... Bikunin has two proteinase inhibitor domains consists of 143 amino acid residues having mass between 19 and 24 kDa (Xu et al. 1998;Seefeldt et al. 2004). The heavy chains have no inhibitory sites, but have the ability to bind in vivo and in vitro to hyaluronic acid (HA) (Bhanumathy et al. 2002;Lord et al. 2013). Complex associated with heavy chains and bikunin is present in varying concentrations in human plasma. ...
Inter-alpha-trypsin inhibitor heavy chain (ITIH4) is IL-6-dependent acute phase protein. Increased expression of ITIH4 during acute phase inflammation of Rheumatoid Arthritis (RA) was reported earlier. Analyzing the heavy chain would, therefore, be crucial to understand the regulation and localisation of Serum-derived Hyaluronic acid-Associated Protein (SHAP) complex in acute phase inflamed conditions. In our previous studies, we have computationally constructed the 3D model of ITIH4 and checked its relevance. But further detailed structural annotation of ITIH4 is required to reveal its surface properties, domain characterisation, and molecular level interaction including drug designing. In the present study, we have utilized our constructed high-resolution three-dimensional (3D) model of ITIH4 protein to reveal the associated surface parameters and its functional role based on the structure-based homology search. Signal peptide prediction has been carried out by the Signalp 4.1 server, whereas COFACTOR server has been utilized for the prediction of active site residues, ligand binding residues, and gene ontology and enzyme classification. Immune epitope database (IEDB) analysis resource was used for the identification of antigenic peptides and Disease-Susceptibility-based SAV Phenotype Prediction (SUSPECT) server revealed the mutation prone residues of ITIH4. Present study indicated that mutation L304H and L304P may enable the protein model highly unstable. The molecular dynamic simulation was carried out for a comparative analysis between native and mutant protein. Additionally, interaction studies implicate that ITIH4 may modulate T cell-mediated inflammation in synovial region during RA. In-silico construction of ITIH4 mutants with single amino acid change may prove to be a tool in experimental studies to understand the importance of each functional domain.
