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Phylogenetic origins of antibody structure. I. Multichain structure of immunoglobulins in the smooth dogfish (Mustelus canis)
Abstract
The elasmobranch Mustelus canis has been shown to produce antibodies to Limulus hemocyanin. The serum of both normal and immunized M. canis contains immunoglobulins having sedimentation coefficients of approximately 7S and 17S. Antibody activity was found in the 17S immunoglobulin which may be dissociated to 7S components with concomitant loss of activity. Both 17S and 7S serum, immunoglobulins were antigenically identical. They consisted of light and heavy chains present in amounts comparable to those of higher vertebrates. Peptide maps indicated that the light chains had an entirely different primary structure than the heavy chains, but that the corresponding chains of 7S and 17S dogfish serum immunoglobulins were similar in primary structure. The heavy chains appeared to resemble the n chains of immunoglobulins of higher vertebrates in their starch gel electrophoretic behavior. It is suggested that the elasmobranch M. canis may have only one major class of immunoglobulins resembling that of macroglobulins (γM-immunoglobulins) seen in higher vertebrates.
The results indicate that the multichain structure of antibodies is an ancient evolutionary development.
... Sharks, members of the elasmobranch class of vertebrates which also includes the skates and rays, are among the most ancient vertebrates for which direct evidence of Ig exists. The first detailed analysis of nonmammalian Ig was performed in a shark, the smooth dogfish [53,55] and these studies demonstrated that antibodies in sharks resemble mammalian IgM with respect to the overall structure, including possession of light chains and Il heavy chains, and physicochemical properties. In addition, sharks were the earliest form of vertebrates for which Ig gene sequence was obtained [56]. ...
... The first detailed analysis of nonmammalian Ig was performed in a shark, the smooth dogfish (Mustelus canis), in 1965 by Marchalonis and Edelman [53,55]. Soon afterward, the structure of Ig from a variety of sharks such as the leopard shark [92], nurse shark [93], and lemon shark [94] was reported. ...
... Soon afterward, the structure of Ig from a variety of sharks such as the leopard shark [92], nurse shark [93], and lemon shark [94] was reported. It was demonstrated that the circulating antibodies in sharks exist in two forms, a 17-19S form of -900 kDa and a 7S form of -180 kDa [55,[92][93][94][95]. These two molecules are antigenically identical and, moreover, resemble mammalian IgM with respect to molecular weight, chain structure, charge dispersity, and carbohydrate content [53,55,[92][93][94][95]. ...
... Limulus hemocyanin was prepared from hemolymph by zone electrophoresis on starch as previously reported (1). Qualitative analyses for precipitins were carried out using test tubes as well as microcapillary tubes according to the procedure described by Kabat and Mayer (11). ...
... Immunodectropkoresis and Double Diffusion in Agar.--These procedurcs and the immunization schedule for rabbits have been previously described (1). ...
The sea lamprey, Petromyzon marinus, has been found to produce specific antibodies after immunization with bacteriophage f2. Antibody activity is localized in 6.6S and 14S fractions of lamprey serum. The 6.6S antibodies were purified by a combination of zone electrophoresis, ion exchange chromatography, and gel filtration. Antigenic analysis of the 6.6S antibodies showed them to be free of other serum proteins and antigenically similar or identical to the 14S fraction. Evidence has been obtained which suggests that the 6.6S immunoglobulins consist of light components (molecular weight 25,000) and heavy components (molecular weight 70,000). In the immunoglobulin, these polypeptides appear to be linked via weak interactions but not by interchain disulfide bonds. Molecular weight analyses support the view that the chains can undergo concentration-dependent dissociation in aqueous solutions. Amino acid analyses showed that the compositions of the light and heavy components were similar and that aspartic acid or asparagine was the predominant amino terminal residue. Starch gel electrophoresis indicated that the subunits of lamprey antibodies are diffusely heterogeneous. The heavy chain mobility corresponded to that of µ-chains and resembled that of heavy chains of shark and sting ray immunoglobulins. In the course of the fractionation a 46S natural hemagglutinin composed of lower molecular weight subunits was isolated. This hemagglutinin did not resemble the lamprey immunoglobulin although it had a similar zone electrophoretic mobility in the β-region. These studies are consistent with the hypothesis that µ-chains were the earliest of the heavy chain classes to emerge and further support the view that the multichain structure of immunoglobulins is a fundamental feature of antibody molecules.
... Some of this early work also suggested a secondary enrichment of 19S. Taken together this was the first evidence that sharks had the capacity to respond to foreign challenge in a coordinated antibodydependent fashion [9][10][11][12][13][14]. In line with classical B cell receptor systems, shark IgM exists in both transmembrane (for activation of lymphocytes) and secretory forms (to bind antigen and induce effector functions of the humoral system) [15]. ...
At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest) antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.
... The fact that 3'M immunoglobulin occurs on the surface of lymphocytes raises an interesting evolutionary possibility. This class of immunoglobulin, expressed as both 19S and 7S forms, arose early in vertebrate phylogeny (33)(34)(35) and statistical evidence suggests that the /x-chain has had a conservative evolutionary history (36). A strong selective factor in maintaining this ancient immunoglobulin molecule may have arisen from its probable role as a surface receptor for antigen. ...
Immunoglobulins were isolated from the surfaces of lymphocytes from a variety of lymphocyte populations including murine and human thymus lymphocytes and murine spleen and thoracic duct lymphocytes. Cell surface proteins were labeled with iodide-¹²⁵I by lactoperoxidase-catalyzed iodination, and recovered in solution either by solubilization in dissociating solvents or active metabolic release. Immunoglobulins were identified and isolated by immunological coprecipitation. The polypeptide chain structure of immunoglobulins isolated from lymphocyte surfaces was analyzed by polyacrylamide gel electrophoresis of reduced, alkylated samples in acid urea. Human and murine thymus lymphocytes possessed only IgM immunoglobulin on their surfaces. This protein contained light chains and µ-type heavy chains and was characterized by a molecular weight of approximately 200,000. Murine splenic lymphocytes from CBA x C57 animals and congenitally athymic (nu/nu) mice possessed both IgM and IgG on their surfaces. The ratio of µ-chain to γ-chain was about 3/1. The presence of IgM on thymus lymphocytes probably does not reflect trace contamination by B lymphocytes because comparable quantities of IgM were isolated from both cell populations. Metabolic turnover data suggest that this immunoglobulin is synthesized by the cell population studied.
These results provide direct evidence for the presence of immunoglobulins composed of light and heavy polypeptide chains on the surfaces of lymphocytes of all classes.
... Poikilothermic vertebrates differ markedly in their immune responses from eutherian mammals in that they generally have only one class of immunoglobulin and seem incapable of a true secondary response (Marchalonis and Edelman 1965;. The frog (Rana catesbeiana) is the most primitive vertebrate which has been shown to have two well-defined classes of immunoglobulin (Marchalonis and Edelman, 1966). ...
Recent work on the immune responses of marsupials is reviewed, with emphasis on cellular immune responses of the macropod marsupial Setonix brachyurus (quokka). Adult marsupials show immunological responses broadly comparable to those of eutherian mammals. However, two immunological mechanisms have evolved to protect the immunologically immature neonate: namely, the passive transfer of antibody and the rapid maturation of immune competence. Thymectomy in marsupials causes a marked lymphocytic depletion of the lymph nodes and spleen, and, in the quokka, a transient abolition of the humoral immune response to SRBC and depression of the response to other antigens. The blood leucocyte response to phytohemagglutinin (PHA) in vitro is also depressed. Although a wasting syndrome does not occur in the quokka, neonatally thymectomized animals have a markedly reduced lifespan. Thymus grafts in either intact or neonatally thymectomized pouch young persist, and in the latter case, fully reconstitute the in vitro leucocyte response to PHA. However, a concomitant restoration of the hemolytic antibody response was not achieved. A high level of lymphocyte chimaerism was found in the grafted animals. These results are discussed with reference to the specificity of cell-cell cooperation in the antibody response, and the possible implication of thymic humoral factors.
... This rapid and straight-forward library construction is possible because of the simple single-domain, molecular architecture of the VNARs, and extends beyond immunized library construction to VNAR phage display libraries derived from naïve and synthetic platforms [70][71][72][73][74][75][76][77]. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity even in mammals [78][79][80][81]. Phage display technology was crucial in elucidating the role of IgNARs in adaptive immunity. ...
Background:
Phage display technology has revolutionized the science of drug discovery by transforming the generation and manipulation of ligands, such as antibody fragments, enzymes, and peptides. The basis of this technology is the expression of recombinant proteins or peptides fused to a phage coat protein, and subsequent isolation of ligands based on a variety of catalytic, physicochemical/binding kinetic and/or biological characteristics. An incredible number of diagnostic and therapeutic domains have been successfully isolated using phage display technology. The variable domain of the New Antigen Receptors (VNAR) found in cartilaginous fish, is also amenable to phage display selection. Whilst not an antibody, VNARs are unquestionable the oldest (450 million years), and smallest antigen binding, single-domains so far identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established, enhancing our understanding of the evolutionary origins of humoral immunity and the unusual but divergent ancestry of the VNARs themselves. VNARs exhibit remarkable physicochemical properties, such as small size, stability in extreme conditions, solubility, molecular flexibility, high affinity and selectivity for target. The purpose of this review is to illustrate the important role phage display has played in the isolation and characterization of potent therapeutic and diagnostic VNAR domains.
... Other properties, such as high carbohydrate content, amino acid composition, five-armed structure as visualized by electron microscopy, and relatively high degree of [3-pleated sheet structure as assessed by circular dichroic spectroscopy (Frommel et al., 1971;Litman et ai., 1971a,b), all lend support to the conclusion that the chondrichthean Ig can justifiably be called an IgM. The monomeric and polymeric forms of IgM in the shark have been shown to be identical by antigenic analysis and biochemical criteria such as amino acid composition and peptide mapping (Marchalonis and Edelman, 1965). It has been suggested that the monomeric IgM in the shark does not represent a precursor or breakdown product of the pentameric molecule; also suggested is that the monomer may carry out some of the physiological reactions of the distinct lower-molecularweight Ig classes seen in the anuran amphibians, reptiles, and mammals (see below). ...
The ability to discriminate self from non-self is fundamental to the organization and survival of metazoan life. This ability resides primarily with the individual cells, although it can be mediated also by specific products secreted by the cells, as will be discussed later. Self/non-self discrimination is essential for the orderly processes of development, differentiation, and organogenesis; in addition, it is required so that an animal can maintain its integrity and prevent invasion and damage by foreign entities, either animate (viruses, bacteria, fungi, metazoan parasites) or inanimate (for example, bacterial exotonins).
... Our understanding of the cartilaginous fish immune system has increased considerably since the first functional studies were performed in the 1960s (Clem and Small 1967;Marchalonis and Edelman 1965;Day et al. 1970). The development of polymerase chain reaction and related molecular techniques enabled the sequencing of individual cartilaginous fish immune genes and/or transcripts, primarily by homology-based cloning. ...
The cartilaginous fishes (sharks, skates, rays, and chimaeras) hold a key evolutionary position, being the most distant group to mammals that possesses a “mammalian-like” adaptive immune system based on immunoglobulins (Ig) and T cell receptors (TCRs), which are somatically rearranged by recombination-activating gene (RAG) proteins, as well as polymorphic/polygenic major histocompatibility complex (MHC) molecules. Cartilaginous fishes are therefore an important research model to investigate the evolution of adaptive immunity and its interplay with the innate system. Despite this, cartilaginous fishes have historically been understudied; while early functional studies revealed sharks were able to produce a humoral response following immune stimulation, subsequent progress was hampered by bottlenecks in immune gene sequencing and a paucity of research tools (such as cell lines and monoclonal antibodies) for use in functional studies.
... 32 According to contemporary tenets, cartilaginous fishes are considered to be the most primitive vertebrates with immunoglobulin (Ig). [36][37][38][39] More recently, Flajnik 40 has proposed that elasmobranchs are the oldest group of vertebrates with an adaptive immune system based on Ig/TCR/MHC. They have been shown to have not only IgM but at least two other isotypes, IgNAR and IgW. ...
The electric ray (Torpedo Marmorata Risso) provides an animal model for the detection of early intraembryonic hemopoietic stem cells in sea vertebrates. The spleen of this bone-marrowless vertebrate appears to be the major site of hemopoietic stem cell differentiation during development and in adulthood. Splenic development in this species was investigated and hemopoietic stem cells were detected in this organ by immunocytochemistry utilizing CD34 and CD38 antibodies. At stage I (2-cm-long embryos with external gills), the spleen contains only mesenchymal cells. At stage II (3-4 cm-long embryos with a discoidal shape and internal gills), an initial red pulp was observed in the spleen, without immunostained cells. At stage III (10-11-cm-long embryos), the spleen contained well-developed white pulp, red pulp and ellipsoids. Image analysis at stage III showed four cell populations, i.e. CD34+/CD38-, CD34+/CD38+, CD34-/CD38+, and CD34-/CD38- cells. The present findings, obtained from an elasmobranch, indicate that the CD34 and CD38 phenotypes are conserved through vertebrate evolution.
... Several studies have demonstrated that Ag-specific VLRB proteins are produced in lamprey in response to particular Ags, such as Brucella abortus, sheep RBCs, Bacillus anthracis, and erythrocytes, and secreted VLRB proteins have a similar function to Abs in jawed vertebrates (4,(15)(16)(17)(18)(19)(20). It has been shown that Ag-specific agglutinins and immunological memory can be generated by the adaptive immune system of lamprey (15,21,22). However, only a few studies have been conducted on hagfish VLRB with respect to its structure and functional similarities with lamprey. ...
The variable lymphocyte receptor (VLR) mediates the humoral immune response in jawless vertebrates, including lamprey (Petromyzon marinus) and hagfish (Eptatretus burgeri). Hagfish VLRBs are composed of leucine-rich repeat (LRR) modules, conjugated with a superhydrophobic C-terminal tail, which contributes to low levels of expression in recombinant protein technology. In this study, we screened Ag-specific VLRBs from hagfish immunized with nervous necrosis virus (NNV). The artificially multimerized form of VLRB was constructed using a mammalian expression system. To enhance the level of expression of the Ag-specific VLRB, mutagenesis of the VLRB was achieved in vitro through domain swapping of the LRR C-terminal cap and variable LRR module. The mutant VLRB obtained, with high expression and secretion levels, was able to specifically recognize purified and progeny NNV, and the Ag binding ability of this mutant was increased by at least 250-fold to that of the nonmutant VLRB. Furthermore, preincubation of the Ag-specific VLRB with NNV reduced the infectivity of NNV in E11 cells in vitro, and in vivo experiment. Our results suggest that the newly developed Ag-specific VLRB has the potential to be used as diagnostic and therapeutic reagents for NNV infections in fish.
... However, even at the time these studies were performed, concerns were raised about the experimental designspecifically, that antigen-specific antibody from the primary response was still evident when the animals were stimulated to examine memory, and that this may have confounded the experimental result (Sigel and Clem, 1966 and disscussion notes therein). Further, sharks were shown to have IgM at high serum concentrations (> 20 mg/mL) but were presumed to possess no other Ig isotypes (Marchalonis and Edelman, 1965). Subsequent work in nurse sharks (Ginglymostoma cirratum) showed that their humoral response is dominated by two Ig isotypes; the first, IgM, is found in both a pentameric (pIgM) and monomeric (mIgM) form. ...
Immunologic memory provides long-term protection against pathogen re-infection and is the foundation for successful vaccination. We have previously shown an antigen-specific recall response in nurse sharks almost one year after primary exposure. Herein, we extend the time between prime and successful recall to >8 years, the longest period for which immunologic memory has been shown in any non-mammalian vertebrate. We confirm that antigen binding is mediated by monomeric IgM and IgNAR, but not pentameric IgM, in both the primary and recall phases. Our inability to find target-binding clones in recombinant VNAR expression libraries suggests that, at least in this instance, antigen-specific memory cells comprise a small fraction of the IgNAR-positive B cells in epigonal and spleen. Further, that the few memory cells present can generate a robust antigen-specific IgNAR titer following re-stimulation. Our results continue to challenge the long-held, but erroneous, belief that the shark adaptive immune system is ‘primitive’ when compared to that of mammals.
... Sharks appear to have only 1 class of Ig, i.e. IgM, but a molecular weight shift from 19s pentameric to a ?S monomeric form of IgM is thought to occur during the immune response (Marchalonis & Edelman 1965, Clem & Small 1967. Recently, some elasmobranchs have been reported to contain a second class of Ig other than IgM (Kobayashi et al. 1984, Kobayashi & Tonionaga 1988) having more than 2 distinctly different heavy chain isotype genes (Kokubu et al. 1988, Harding & Arnemiya 1990). ...
Antisera obtained from flounder Paralichthys olivaceus immunized with Edwardsleila tarda were fractionated using Sephacryl S-300 gel filtration. The flounder was found to contain various sizes of immunoglobulins (Igs) ranging from <200 to >700 kDa. These Igs were combined into 3 pools according to molecular weight; pool I consisted of high molecular weight (HMW) Igs of >700 kDa, pools II and III consisted of low molecular weight (LMW) Igs of 230 to 700 kDa (pool II) and <230 kDa (pool III). After being further purified by hydroxyapatite column chromatography and isoelectric focusing, HMW Ig in pool I and LMW Ig in pool III were compared with each other by SDS-PAGE under reducing conditions. They were found to be composed of 1 size of heavy chain and 2 sizes of light chain. While HMW Ig in pool I contained 68 kDa H chains and 22 kDa and 24 kDa L chains, LMW Ig in pool III contained 69 kDa H chains and 22 kDa and 26 kDa L chains. A set of 5 monoclonal antibodies (MAbs) against HMW Igs was prepared. The reactivities of these MAbs with Igs in the 3 pools were determined by enzyme linked immunosorbent assay (ELISA) and dot immunoassay. All 5 MAbs reacted strongly only to the HMW Igs. Results suggest that flounder lgs comprise LMW lgs as well as HMW Igs and the physicochemical and antigenical characteristics of LMW Igs are different from those of HMW Igs.
... Despite 450 million years of divergence from mammals, cartilaginous fish have both TM and secretory forms of IgM H chains [12,13]. Besides the pentameric form of secreted IgM classically called '19S,' shark B cells also secrete a monomeric form, classically '7S' [14][15][16][17][18]. 7S IgM is thought to be a mammalian IgG equivalent, both with respect to its ability to penetrate extravascular spaces and its enhanced binding strength late in adaptive immune responses, whereas previous work also suggested that 19S IgM does not participate in antigen-specific responses [19,20]. ...
Blimp-1 is the master regulator of plasma cell development, controlling genes such as those encoding J-chain and secretory Ig heavy chain. However, some mammalian plasma cells do not express J-chain, and mammalian B1 cells secrete "natural" IgM antibodies without upregulating Blimp-1. While these results have been controversial in mammalian systems, here we describe subsets of normally occurring Blimp-1(-) antibody-secreting cells in nurse sharks, found in lymphoid tissues at all ontogenic stages. Sharks naturally produce large amounts of both pentameric (classically '19S') and monomeric (classically '7S') IgM, the latter an indicator of adaptive immunity. Consistent with the mammalian paradigm, shark Blimp-1 is expressed in splenic 7S IgM-secreting cells, though rarely detected in the J-chain(+) cells producing 19S IgM. Although IgM transcript levels are lower in J-chain(+) cells, these cells nevertheless secrete 19S IgM in the absence of Blimp-1, as demonstrated by ELISPOT and metabolic labeling. Additionally, cells in the shark bone marrow equivalent (epigonal) are Blimp-1(-) . Our data suggest that, in sharks, 19S-secreting cells and other secreting memory B cells in the epigonal are maintained for long periods without Blimp-1, but like in mammals, Blimp-1 is required for terminating the B-cell program following an adaptive immune response in the spleen. This article is protected by copyright. All rights reserved.
1.1. A detailed electrophoretic study was made of different developmental stages of the bull shark, Carcharhinus leucas Müller and Henle, 1841.2.2. Both qualitative and quantitative variations were found to exist between newborn and adult bull sharks.3.3. Variations in the globulin portion may be related to the development of immunoglobulins.
A unique "monoclonal" gamma globulin was detected in the serum of a patient with malignant disease of the plasma cell-lymphatic system. This protein had antigenic determinants common to 19S γM-globulin (macroglobulin) but there were marked physicochemical differences. The clinical and pathologic findings in this patient differed from those usually found in patients with multiple myeloma or Waldenström's macroglobulinemia. Evidence obtained from density gradient and gel filtration studies of whole serum indicated that the γM antigenic determinants were located primarily on molecules smaller in size than 19S-globulin. The anomalous gamma globulin, isolated from serum, had a sedimentation coefficient (s20, w) of 6.5S, consisted of μ-type heavy chains and λ-type light chains and differed slightly from the monomeric subunits of a reduced alkylated 19S γM-globulin. This protein may be related to low molecular weight γM antibodies previously described in a normal serum and in several disease states.
The nature of fish antibodies (concentrating primarily on the most studied species of bony and cartilaginous fishes) is discussed in terms of their immunoglobulin biochemistry and immunobiology. The major serum immunoglobulin (IgM) is described in detail, and structural variants of IgM are discussed in terms of their distribution in different fish species, and different anatomical sites within a fish (e.g. blood, mucus, bile). Structural variation in IgM includes the size of the constituent heavy and light polypeptide chains, and the extent to which they are covalently associated with one another. The intramolecular heterogeneity of binding sites for antigen on IgM is reviewed and possible mechanisms for the phenomenon are presented. The evidence suggests that some, but not all, species of fish possess a detectable J chain in their IgM. The general nature of the fish immune response is that IgM antibody of moderate affinity is produced and prolonged or repeated immunization: (a) fails to produce a switch to production of a non-IgM class of antibody, and (b) fails to induce substantial increases in the affinity of the specific antibodies. Evidence supports a conclusion that fish lack the typical secondary antibody response seen in mammals, and possess antibodies of limited heterogeneity. Our current understanding of the genetic basis for fish antibodies is presented. Fish appear to utilize the same basic genetic elements as mammals to encode and regulate the expression of their immunoglobulins. The teleost heavy chain (IgH) locus resembles that of mammals and amphibia in its organization. The IgH locus of elasmobranchs is arranged in a unique multicluster organization. The light chain loci of elasmobranchs are organized analogously to the heavy chain locus (in multiclusters). The structure of the light chain locus of teleosts is presently unknown. Teleost fish utilize a unique pattern of RNA processing to generate the secreted and membrane receptor forms of the IgM heavy chain. The genes encoding the unique low molecular weight Ig heavy chain found in skates and rays have been cloned and sequenced, and also display the multicluster pattern of organization. Teleost fish appear to have normal numbers of variable regions: it is hypothesized (but as yet unproven) that the failure of their IgM to increase in affinity is due to a deficiency of somatic hypermutational mechanisms in their Ig gene variable regions during B lymphocyte differentiation.
Allotypic markers originally found on rabbit IgG have been shown to reside also on both the heavy and light chains of rabbit IgM. The light chains of IgG and IgM are the same, but the heavy chains of these two immunoglobulins differ extensively. The extent to which the allotypes present on the heavy chains are duplicated on the two immunoglobulins has been investigated by inhibition of phage neutralization and by a radiotracer technique. The inhibition by anti-allotype sera of phage neutralization by IgM antibody was shown to be essentially completely removed by prior absorption of the anti-allotype sera with IgG of the same heavy chain allotype. A preparation of IgM containing allotype a1 exhausted extensively but not completely, the precipitating ability of anti-a1 serum for a1, b4 IgG labeled with I125. It is concluded that in the anti-allotype sera investigated, essentially all of the antibodies directed against the allotypic sites present on IgM react with IgG, but not all the antibodies directed against the allotypic sites present on IgG are capable of reacting with IgM. The implications of these findings with regard to the genetic control of immunoglobulin synthesis and the evolution of the immunoglobulin genome are discussed.
The proteins on surfaces of living splenic lymphocytes from normal BALB/c mice were iodinated enzymatically. Such cells were
fractionated into two sub-populations: one composed almost exclusively of small lymphocytes and the other mainly of large
lymphocytes and plasma cells. Specific immunoprecipitation of radiolabeled surface Ig obtained from lysates of these cell
populations indicated that approximately 2–3% of the acid-precipitable radioactivity from the cell surface is Ig. Moreover,
95% of the H chain radioactivity from the Ig of the small lymphocyte fraction and 90% from the large lymphocyte-plasma cell
fraction was characterized as µ by precipitation with anti-µ sera as well as by molecular weight determination on polyacrylamide
gels in sodium dodecyl sulfate. The Ig was recovered from the cell surface in the form of an IgM monomer. Control experiments
suggested that the monomer did not result from depolymerization of 19S IgM by the methods used to radiolabel and isolate the
molecule. 3H-tyrosine labeling of IgM produced by meyloma cells and radio-iodination of IgM in solution gave the same ratios of µL radioactivity
as radiolabeling of IgM on cells, indicating that the tyrosine residues of L and µ-chains of cell surface IgM are available
to the lactoperoxidase during the iodination. This is consistent with the hypothesis that cell surface IgM is entirely on
the outside of the plasma membrane presumably attached to it by its Fc fragment. These results, together with previous reports
by others, suggest that IgM, in its monomeric form, is the main antigen-specific receptor on lymphocytes of normal mice.
The natural hemagglutinin of the oyster Crassostrea virginica was examined for possible structural relationship with vertebrate immunoglobulins. The hemagglutinin activity was associated
with a heterogeneous group of rapidly sedimenting molecules. The major active component in oyster hemolymph has a sedimentation
coefficient of 33.4 S, but minor components with lower sedimentation coefficients are also present. The molecules can be dissociated
into subunits consisting of single polypeptide chains having an approximate molecular weight of 20,000. Complete dissociation
could be observed in 5 m guanidine-HCl indicating noncovalent linkage of the subunit. Amino acid composition of the subunit was distinguished by a
relatively high histidine content and the absence of lysine. The sequence of the first 2 residues from the NH2 terminus of the subunit was found to be Thr-Ala. Mannose, galactose, and glucosamine were present with a total carbohydrate
content of 13%. These data do not demonstrate obvious structural similarities to mammalian immunoglobulins.
The literature relating to the immune response of fish has been reviewed. Non-specific immune mechanisms similar to those of other vertebrate classes occur in fish. Similarly specific cell-mediated immunity has been demonstrated at all levels of evolution, from the cyclostomes to the teleosts. A humoral antibody system also occurs in all classes offish but varies considerably in relation to phylogenetic status. In the cyclostomes, only immuno-proteins with properties intermediate between the immunoglobulms of vertebrates and the non-specific agglutinins and lysins of invertebrates have been demonstrated. In the elasmo-branchs and chondrosteans 7 and 19s irnmunoglobulins of IgM type occur. In holosteans the 19s form is predominant whereas in teleosts, 7 and 19s forms occur, with some evidence of specialization in the 7s form. In the phylogenetically most advanced fish, the Dipnoi, two immunoglobulin classes, structurally analogous to IgM and IgG, have been described.
A characteristic feature of both cell-mediated and antibody mediated immune responses in fish is their dependence upon environmental temperature. There is also evidence that, in some species at least, nutritional factors and behaviour patterns may also influence the immune response.
Attempts at artificial immunization of fish against infectious disease have met with varied success. It is probable that better results could be achieved with live vaccine strains, particularly if applied under conditions optimal for the immune responses.
Natural‘antibodies’are substances found in the blood of animals that have not been immunised against infective agents. However, exposure to these agents or to cross-reacting antigens may well have taken place. Fish contain naturally-occurring, relatively nonspecific, lectin-like proteins or glycoproteins, which are distinct from immunoglobulins, and which react with a wide variety of antigens and may confer some degree of immunity against natural infection. In most cases the cause of the antigenic stimulus is not obvious although the formation of these‘antibodies’may have been brought about by exposure to various micro-organisms. Many of these antibody-like molecules behave in a similar manner to immune antibodies or immunoglobulins and cross-react with specific carbohydrate moieties on the cell walls of bacteria, erythrocytes and certain other cellular antigens, due to the presence of similar antigenic determinants.
Low molecular weight (LMW) IgM, the monomeric subunit of pentameric IgM, has been detected in the sera of patients with a wide variety of rheumatic, infective, and lymphoproliferative diseases but is not found in healthy individuals. The factors concerned in the production of LMW IgM are largely unknown. Four hypotheses have been suggested to explain its presence. First, in vivo breakdown of immune complex-bound pentameric IgM may release monomeric IgM. This could possibly explain the frequent occurrence of LMW IgM in immune complex states. Second, a defect during IgM polymerization could result in the liberation of monomeric IgM into the circulation. This is the favored postulate in B cell lymphoproliferative disorders associated with IgM paraproteins. Third, LMW IgM exists in small quantities as a membrane-bound immunoglobulin on certain B cells, and its release into the circulation could theoretically occur during rapid cell proliferation. However, membrane-bound LMW IgM has different physiochemical properties and low synthetic rates compared with secretory pentameric IgM, and its release into the circulation in quantities comparable with those found is not considered possible. Last, the presence of LMW IgM may represent a phylogenetic reversion of the immune response, as LMW IgM exists naturally in lower vertebrates. This may explain its occurrence in a number of chronic immune-stimulated states such as rheumatoid arthritis (RA) and chronic infective disorders and its close association with rheumatoid factor (RF), which is also frequently found in these conditions. This study explored the second and fourth of these hypotheses. We have documented the active secretion of LMW IgM by pokeweed-stimulated peripheral blood mononuclear cells (PBMC) obtained from RA patients in vitro, but not from healthy control subjects. A correlation was found between the levels of LMW IgM synthesized in vitro and circulating LMW IgM.
Low molecular weight (LMW) IgM was measured in the serum and synovial fluid of patients with rheumatoid arthritis (RA) and other rheumatic diseases. High levels were seen in RA, particularly in rheumatoid vasculitis and Felty's syndrome, and significant correlations occurred between LMW IgM and the rheumatoid factor (RF) level and other indices that reflected active or severe disease. LMW IgM-RF, measured by radio-immunoassay in those column fractions containing LMW IgM, correlated significantly with LMW IgM (P < 0.005); preliminary experiments suggested that in some sera, a considerable proportion of the LMW IgM consisted of LMW IgM-RF. We conclude that LMW IgM and LMW IgM-RF may have important implications in the immunopathogenesis of RA and other rheumatic diseases.
1.1. A method for purification of β1-globulin from dogfish (Scyllium stellare) serum is described, involving precipitation with ammonium sulphate, chromatography on DEAE-cellulose and preparative electrophoresis on a Pevikon block.2.2. The purified protein is not perchlorosoluble, although it has a carbohydrate content of about 14 per cent with galactose, mannose, glucose, glucosamine, galactosamine, fucose and sialic acid. It has a sedimentation constant of 4·28 S. This glycoprotein forms with hemin a red-coloured complex.3.3. It is suggested that this protein henceforth be called “dogfish hemophilin”.
Cartilaginous fish are the oldest vertebrate class possessing an adaptive immune system typified by major histocompatibility complex (MHC) T cell receptor and immunoglobulin (Ig). Three heavy chain classes have been identified in cartilaginous fish, bona fide IgM, IgNAR, thought to be important for secondary responses, and IgW. In most vertebrates the immune repertoire expressed early in development has innate features, such as antibody variable regions in germline configuration with little somatic modification, produced by a specialized B lymphocyte subset. We have identified a fourth class of Ig, IgMinnate (in) in the nurse shark (Ginglymostoma cirratum ) expressed preferentially in neonatal primary and secondary lymphoid tissues with a germline, (germline-joined) non-diverse variable region, and at maturity expressed only in the epigonal organ, the bone marrow equivalent. IgMin heavy chain associates covalently with light chains and is most similar in sequence to pH chains, but like mammalian IgG has only three constant domains; deletion of the ancestral IgM C2 domain defines both IgG and IgMin, demonstrating molecular convergence.The adult nurse shark spleen has well-defined, well-vascularized white pulps populated by a central "T cell" zone filled with a network of dendritic cells (DC) and rare plasma cells, surrounded by a B cell zone containing IgM+ or IgNAR+ and MHC class II + B cells. Newborn splenic white pulp matures into B and T cell zones with a delay of MHC class II expression on B cells, and delay in T cell and DC movement into the white pulp, all comparable to mammalian development. IgNAR+ B cells are also delayed in development, concurrent with arrival of T cells and DCs. The neonatal epigonal organ is the major site of B lymphopoiesis based on the presence of transmembrane IgM, developing B cells, and RAG1 and TdT expression.Shark immunization studies established the antigen was transported into the splenic red pulp by macrophages after one week and by four weeks was moved into the white pulp "T cell" zone and presented on the cell surface of putative DCs. Clusters of IgNAR+ secretory cells in the same "T cell" zones are hypothesized to be sites of somatic mutation and selection, perhaps analogous to mammalian germinal centers.
SYNOPSIS. Recent evidence suggests Ihat amphibians represent a key transition stage in the evolution of immunological competence. In particular, anuran amphibians exhibit a significant advance relative to more primitive vertebrates in their capacity to synthesize two distinct classes of immunoglobulins in response to antigenic stimulation.These two classes of antibody molecule resemble the γM and γG antibodies of man in size and polypeptide chain structure. Furthermore, cellular aspects of antibodyproduction in anura resemble those of mammals in the method of retention of antigen and the range of antibody-forming cells (lymphocytes to plasma cells).
In addition to these parameters of antibody-formation, a state of specific nonresponsiveness (high zone tolerance) can be induced in adult anura by pretreatment with large doses of protein antigen.
Studies of larval anurans establish that these forms become immunologically competent early in development, but possess only γM immunoglobulins. These larval stages provide feasible model systems for the study of the activation of genes which specify immunoglobulins.
Antibody-formation in urodeles is discussed in relation to the emergence of immunological competence in amphibians.
This chapter acquaints individuals interested in fishes with the basic aspects of the biology and evolution of antibody synthesis and associated phenomena and to show in some detail how the study of fish relates to this area. As the point of view is that of comparative immunology, a brief history of this subject is considered first. Emphasis is placed on the aspects of the physiology of comparative immunology rather than on the problems relating to the identification of subpopulations. The discussion included is written with the assumption that the prime concern is the observations and concepts and follows through the literature those citations of methods that are of specific interest. The chapter provides a general concept from the work on mammalian antibodies, especially human, and to show how the study of fishes is assisting in enlarging this concept, which is in a state of rapid evolution. It outlines the complex nature of the immunoglobulins. Obviously, the mechanism for the synthesis of these molecules is well-evolved in mammals; and it is of prime interest to understand its evolutionary origins and phylogeny.
Giant grouper serum was shown to contain 16 S (∼3 mg per ml of serum) and 6.4 S (∼6 mg per ml of serum) immunoglobulins. The 16 S immunoglobulin had a molecular weight of ∼700,000, a relatively high hexose content, and was composed of approximately equimolar amounts of H and L polypeptide chains. It is suggested that this molecule resembles immunoglobulin M on the basis of polypeptide chain properties but that it is most likely a tetramer instead of the "typical" 19 S pentamer of immunoglobulin M. The 6.4 S immunoglobulin appeared structurally to represent a fragment of the 16 S molecule; the major difference being that the 6.4 S H chain was missing ∼30,000 daltons.
In attempting to fit the class Osteichthyes into a scheme of vertebrate immunoglobulin evolution, it was suggested that the "primitive" form of polymeric immunoglobulin M may be a tetramer of subunits held together by both disulfide and noncovalent bonds.
A considerable amount of research has been done on antibody synthesis. This is first and foremost a result of its practical importance, which is reflected in the fact that a considerable portion of the population is vaccinated each year with the most varied antigens. It is also the reason for the comprehensive literature on the productive phase of antibody synthesis, i.e., the period commencing at the moment antibodies appear in the blood. However, at the present, many investigators are concentrating on the elucidation of processes which are initiated directly after administration of antigen to the organism and which ultimately lead to formation of antibody molecules. As in the biosynthesis of other proteins, nucleic acids can be expected to play an important role in the mechanism of antibody formation. Hence, it is not surprising that much attention is being devoted at the present time to synthesis of nucleic acids in antibody-forming cells and tissues.
From the experimental material presented in the foregoing chapters it is evident that many of the chief questions concerning antibody biosynthesis are still unanswered. The undisputably most important of these questions is how the specific configuration of the antibody binding site is formed. Inasmuch as this configuration is, according to current conceptions, associated with the specific structure of the N-terminal parts of peptide chains, it is necessary to elucidate the mechanism responsible for the variability of these chain parts. It should be taken into account that the differences between the variable chain parts are confined to certain chain sections between which are found very stable regions [17, 47–49], It should also be borne in mind that the differences between the variable parts of the chains are more likely to be due to point mutations rather than to other genetic mechanisms. A second important problem is the way in which antigen exerts its effect. At present, the mechanism of action of antigens is not at all understood.
Members of the phylum Echinodermata (around 6000) are the only known species of deuterostome invertebrates. The living echinoderms are at present classified into three subphyla, the Crinozoa, the Asterozoa, and the Echinozoa which comprise the echinoderms commonly known as sea lilies and feather stars, then starfish, sea stars, basket stars, serpent stars and brittle stars, and finally sea urchins, heart urchins, sand dollars, and sea cucumbers (Moore, 1966-1978).
Numerous studies have revealed that the lymphoid tissue of the chicken comprises two distinct peripheral components with basically separate functions. If newly hatched chicks are subjected to complete extirpation of the bursa of Fabricius plus near lethal total body irradiation, these chickens grow up as immunological cripples unable to produce demonstrable amounts of circulating antibodies (1, 2). Such chickens lack both of the known immunoglobulins, IgM and IgG. Nevertheless, even though grossly defective in ability to synthesize antibody and gamma globulin, such chickens have normal or essentially normal ability to develop and express cell-mediated immunity. In contrast, chickens subjected to total body irradiation and complete thymic extirpation grow up as immunological cripples unable to execute the cell-mediated immunities; i.e., delayed allergy, homograft immunity, and capacity to execute the graft-versus-host reactions. This division of immunological functions on the basis of influence of two separate central lymphoid organs is reflected in specialization of structure in the peripheral lymphoid organs; e.g., spleen, lymph nodes, skin, and intestinal lymphoid aggregates. Chickens irradiated and bursectomized in the newly hatched period completely lack plasma cells and germinal centers, but possess normal numbers of circulating small lymphocytes and normal aggregates of lymphocytes in the spleen, lymph nodes and other lymphoid cell organs and tissues. The thymus-dependent lymphocyte population is responsible for the cell-mediated immune reaction while the bursa-dependent germinal centers and plasma cells are responsible for the synthesis of immunoglobulin and antibody.
In mammals, immunologic responses can be divided into those mediated by humoral antibodies (antibody-mediated immunity) and by cells (cell-mediated immunity). Both depend on the activity of small lymphocytes that are themselves ultimately derived from bone marrow precursors.(226) The stem cells, which originate in the early embryonic stage in the yolk sac and in later stages of development in the liver, and in adults in the bone marrow, differentiate to form two distinct lymphocyte populations, one thymus-derived (T lymphocytes), the other bone-marrow-derived (B lymphocytes)(77,167) The life span of lymphocytes is not uniform. One can distinguish two populations of lymphocytes: a short-lived population of small, medium, and large lymphocytes with a life span of 1?10 days, and a long-lived population of small lymphocytes with a life span of some 100 days(275); some of these small lymphocytes have a life span of 10 years and more.(192)
During the past 10 years we have been reinvestigating the question of the phylogenetic development of immunity (Good and Papermaster, 1961, 1964; Papermaster, Condie and Good, 1962; Papermaster et al, 1963; Papermaster et al., 1964, 1964 a; Finstad, Papermaster and Good, 1964; Finstad, 1964, 1966; Finstad and Good, 1964, 1966; Good and Finstad, 1964, 1964 a; Finstad and Fichtelius, 1965; Good et al., 1966; Good, 1966; Fish, Pollara and Good, 1966; Pollara, Finstad and Good, 1966; Good et al., 1966; Gewürz et al., 1966). Invertebrates have been found to have protective mechanisms based on serological reactions (Glaser, 1918; Cantacuzéne, 1923; Bernheimer, 1952; Gushing and Campbell, 1957; Li, 1960; Gushing, 1962) and have been shown in some studies to have reactions thought to be of immunological nature (Phillips, 1960; Phillips and Yardley, 1960, 1960 a; Osawa and Yabuubhi, 1963; Michelson, 1963; Pan, 1963; Bang, 1966). No one has thus far defined in the invertebrates the presence of the adaptive immune responses (Good and Papermaster, 1964; Teague and Friou, 1963, 1964). We chose to investigate this question by first setting down the characteristics which we considered definitive of the mammalian immune responses dependent upon the lymphoid system of cells, and second, attempting to trace the phylogenetic development of these processes and the complex system of organs and cells which make up the lymphoid apparatus. In recent months our studies have come to focus on the germinal centers and the lympho-epithelial organs essential to the development of germinal centers (Good, 1966). Consequently, we consider a brief review of our analysis of the phylogenetic development of the lymphoid system and immunological functions to be germane to the considerations of this Conference.
Congenital varicella are a rare but severe condition. Women should be advised during pregnancy to protect themselves against the infection. Newborn babies of mothers who develop varicella during the last two weeks of gestation or during the first days after delivery are to be considered as being infected. There are reports about 39 infants who acquired the infection in utero with eight fatal cases. The disease seems to be more severe in those babies whose mothers became ill during the last five days before parturition or later.—One case of uncomplicated congenital varicella is reported. The baby had a high level of gamma M-Globulin in its serum which we think to indicate the capacity of immunologic response at even such a low age.
As in mammals, cartilaginous and teleost fishes possess adaptive immune systems based on antigen recognition by immunoglobulins (Ig), T cell receptors (TCR), and major histocompatibility complex molecules (MHC) I and MHC II molecules. Also it is well established that fish B cells and mammalian B cells share many similarities, including Ig gene rearrangements, and production of membrane Ig and secreted Ig forms. This chapter provides an overview of the IgH and IgL chains in cartilaginous and bony fish, including their gene organizations, expression, diversity of their isotypes, and development of the primary repertoire. Furthermore, when possible, we have included summaries of key studies on immune mechanisms such as allelic exclusion, somatic hypermutation, affinity maturation, class switching, and mucosal immune responses.
The lymphoid system and capability for adaptive immune responses evolved in the lower vertebrates. We have previously defined adaptive immune responsiveness as ability to: make specific antibody, show immunologic memory upon repeated injection of antigen, reject homografts of skin or scale, exhibit second-set rejection reaction, and show proliferation of cells involved in the immune response [1, 2, 3, 4, 5].
Im nachfolgenden Beitrag sollen die Vorgänge der Regeneration, Hyperplasie und Cancerisierung am Beispiel des lymphoretikulären Systems besprochen werden. Die Berechtigung zu diesem Vorhaben, wenig mehr als 10 Jahre nach Erscheinen des Handbuchartikels von Masshoff (1955) über „die physiologische Regeneration“, ergibt sich aus dem Umstand, daß in der Zwischenzeit die Erforschung der immunbiologisch aktiven Zellsysteme umwälzende Fortschritte gebracht hat. Eine erneute Bearbeitung dieser Fragen erschien daher unumgänglich. Die Gründe für die rasche Entwicklung auf dem Gebiet der Immunologie, in deren Rahmen das lymphoretikuläre System gehört, liegen vor allem in der Verfeinerung der Untersuchungsmethoden. Es ist heute möglich, Zellen stabil zu markieren und auf diese Weise deren Schicksal zu verfolgen. Wir sind ferner in der Lage, biochemische Vorgänge auf cellulärer Ebene zu verfolgen und makromolekulare Bestandteile des Gewebes aufgrund ihrer Antigeneigenschaften immuno-histochemisch zu erfassen. Diese und weitere Gegebenheiten gestatten es, experimentell belegbare Aussagen zu machen, wo früher nur Vermutungen möglich waren. Wie wir sehen werden, haben die neu gewonnenen Erkenntnisse auch für die Beurteilung von Regeneration, Hyperplasie und Cancerisierung ihre große Bedeutung. Allerdings bleibt hervorzuheben, daß die Geschwindigkeit der heutigen wissenschaftlichen Entwicklung wenig mehr als eine Standortbestimmung zuläßt.
Joining chain (J chain) is a small polypeptide that regulates multimerization of secretory IgM and IgA, the only two mammalian Igs capable of forming multimers. J chain also is required for poly-Ig receptor-mediated transport of these Ig classes across the mucosal epithelium. It is generally assumed that all plasma cells express J chain regardless of expressed isotype, despite the documented presence of J chain(-) plasma cells in mammals, specifically in all monomeric IgA-secreting cells and some IgG-secreting cells. Compared with most other immune molecules, J chain has not been studied extensively, in part because of technical limitations. Even the reported phenotype of the J chain-knockout mouse is often misunderstood or underappreciated. In this short review, we discuss J chain in light of the various proposed models of its expression and regulation, with an added focus on its evolutionary significance, as well as its expression in different B cell lineages/differentiation states.
IgD has been found in almost all jawed vertebrates, including cartilaginous and teleost fish. However, IgD is missing in acipenseriformes, a branch that is evolutionarily positioned between elasmobranchs and teleost fish. Here, by analyzing transcriptome data, we identified a transcriptionally active IgD-encoding gene in the Siberian sturgeon (Acipenser baerii). Phylogenetic analysis indicated that it is orthologous to mammalian IgD and closely related to the IgD of other fish. The lengths of sturgeon membrane-bound IgD transcripts ranged from 1.2 kb to 6.2 kb, encoding 3 to 19 CH domains. As in teleosts, the first CH domain of the sturgeon IgD transcript is also derived from μCH1 by RNA splicing. However, the variable region of the expressed sturgeon IgD shows limited V(D)J usage. In addition to IgD, three IgM variants were also identified in this species, whereas no IgT/Z-encoding genes were observed. This study bridges the gap in Ig evolution between elasmobranchs and teleosts and provides significant insight into the early evolution of immunoglobulins.
The immune system of fish comprises a complex mixture of nonspecific and specific cellular and humoral responses to antigen stimulation that are similar to those of higher vertebrates. The immune system of channel catfish has received a great deal of attention with the emergence of economically important infectious diseases and the need for effective vaccines, and has provided a unique model system for study of the immune systems of lower vertebrates. Research studies have elucidated many components of the catfish immune system, although much remains unknown. Modulation of immune responses in fish in relation to water temperature is a major factor influencing the onset and duration of the immune response. Genome analysis of channel catfish using molecular approaches, such as expressed sequence tag analysis, facilitates identification of important immune response genes. A more comprehensive understanding of immune function in channel catfish is critical for the development of improved methods to manage infectious diseases in catfish aquaculture. Enhancing the immune response through vaccination, improved nutrition, or the addition of immunostimulants to the feed has been a successful strategy with other aquaculture species and will surely be explored further in channel catfish culture. The description of the catfish immune system provided here includes information derived specifically from research on channel catfish, and also includes examples from different teleost species. Where the specific functional component for channel catfish is unknown, a description of a representative teleost immune system is provided.
Teleosts (bony fish) are thought to primarily or exclusively possess a single, structural form of immunoglobulin (Ig), a tetrameric IgM. However, in species wherein intact Ig has been electrophoretically analyzed under denaturing, non-reducing conditions, a significato degree of structural diversity has been revealed. This IgM molecule appears to be assembled with great latitude in the degree of disulfide crosslinking between monomeric or hallmark subunits composing the complete IgM molecule. This heterogeneity in the basic structure (herein referred to as redox forms) is not due to isotopic differences as each B cell produces this heterogeneity within its immunoglobulin product. Additionally, in the case of the catfish, a single fish/mouse chimeric Ig H gene is capable of producing IgM with a comparable amount of structural heterogeneity within the mouse cell. Thus, the piscine B lymphocyte routinely assembles a variety of redox forms from one IgM H chain. This has both profound biosynthetic implications for macromolecular assembly processes as well as intriguing possibilities for the generation of teleost Ig functional diversity.
The procedures for carrying out the hemagglutination and hemagglutination-inhibition reactions with tannic acid-protein treated cells have been described. The advantages, limitations and general applications of these methods have been illustrated experimentally and discussed.
1. The California hagfish, Eptatretus stoutii, seems to be completely lacking in adaptive immunity: it forms no detectable circulating antibody despite intensive stimulation with a range of antigens; it does not show reactivity to old tuberculin following sensitization with BCG; and gives no evidence of homograft immunity.
2. Studies on the sea lamprey, Petromyzon marinus, have been limited to the response to bacteriophage T2 and hemocyanin in small groups of spawning animals. They suggest that the lamprey may have a low degree of immunologic reactivity.
3. One holostean, the bowfin (Amia calva) and the guitarfish (Rhinobatos productus), an elasmobranch, showed a low level of primary response to phage and hemocyanin. The response is slow and antibody levels low. Both the bowfin and the guitarfish showed a vigorous secondary response to phage, but neither showed much enhancement of reactivity to hemocyanin in the secondary response. The bowfin formed precipitating antibody to hemocyanin, but the guitarfish did not. Both hemagglutinating and precipitating antibody to hemocyanin were also observed in the primary response of the black bass.
4. The bowfin was successfully sensitized to Ascaris antigen, and lesions of the delayed type developed after challenge at varying intervals following sensitization.
5. The horned shark (Heterodontus franciscii) regularly cleared hemocyanin from the circulation after both primary and secondary antigenic stimulation, and regularly formed hemagglutinating antibody, but not precipitating antibody, after both primary and secondary stimulation with this antigen. These animals regularly cleared bacteriophage from the circulation after both the primary and secondary stimulation with bacteriophage T2. Significant but small amounts of antibody were produced in a few animals in the primary response, and larger amounts in the responding animals after secondary antigenic stimulation.
6. Studies by starch gel and immunoelectrophoresis show that the hagfish has no bands with mobilities of mammalian gamma globulins; that the lamprey has a single, relatively faint band of this type; and that multiple gamma bands are characteristic of the holostean, elasmobranchs, and teleosts studied. By this method of study, the bowfin appeared to have substantial amounts of gamma2 globulin.
7. We conclude that adaptive immunity and its cellular and humoral correlates developed in the lowest vertebrates, and that a rising level of immunologic reactivity and an increasingly differentiated and complex immunologic mechanism are observed going up the phylogenetic scale from the hagfish, to the lamprey, to the elasmobranchs, to the holosteans, and finally the teleosts.
Ion-exchange adsorbents have been prepared from cellulose under conditions such that physical properties suitable for column chromatography are maintained. These adsorbents possess high capacity for the adsorption of proteins, yet permit elution under mild conditions. Titration curves are presented.
1. The California hagfish, Eptatretus stoutii, seems to be completely lacking in adaptive immunity: it forms no detectable circulating antibody despite intensive stimulation with a range of antigens; it does not show reactivity to old tuberculin following sensitization with BCG; and gives no evidence of homograft immunity. 2. Studies on the sea lamprey, Petromyzon marinus, have been limited to the response to bacteriophage T(2) and hemocyanin in small groups of spawning animals. They suggest that the lamprey may have a low degree of immunologic reactivity. 3. One holostean, the bowfin (Amia calva) and the guitarfish (Rhinobatos productus), an elasmobranch, showed a low level of primary response to phage and hemocyanin. The response is slow and antibody levels low. Both the bowfin and the guitarfish showed a vigorous secondary response to phage, but neither showed much enhancement of reactivity to hemocyanin in the secondary response. The bowfin formed precipitating antibody to hemocyanin, but the guitarfish did not. Both hemagglutinating and precipitating antibody to hemocyanin were also observed in the primary response of the black bass. 4. The bowfin was successfully sensitized to Ascaris antigen, and lesions of the delayed type developed after challenge at varying intervals following sensitization. 5. The horned shark (Heterodontus franciscii) regularly cleared hemocyanin from the circulation after both primary and secondary antigenic stimulation, and regularly formed hemagglutinating antibody, but not precipitating antibody, after both primary and secondary stimulation with this antigen. These animals regularly cleared bacteriophage from the circulation after both the primary and secondary stimulation with bacteriophage T(2). Significant but small amounts of antibody were produced in a few animals in the primary response, and larger amounts in the responding animals after secondary antigenic stimulation. 6. Studies by starch gel and immunoelectrophoresis show that the hagfish has no bands with mobilities of mammalian gamma globulins; that the lamprey has a single, relatively faint band of this type; and that multiple gamma bands are characteristic of the holostean, elasmobranchs, and teleosts studied. By this method of study, the bowfin appeared to have substantial amounts of gamma(2) globulin. 7. We conclude that adaptive immunity and its cellular and humoral correlates developed in the lowest vertebrates, and that a rising level of immunologic reactivity and an increasingly differentiated and complex immunologic mechanism are observed going up the phylogenetic scale from the hagfish, to the lamprey, to the elasmobranchs, to the holosteans, and finally the teleosts.
The immune response of turtles to giant keyhole limpet hemocyanin was studied by means of the precipitation of I*-hemocyanin by antiserum. The pertinent findings were:
1. The 18 S protein constitutes 13 to 26% of the total normal serum protein in the turtle.
2. In an antibody response rapidly sedimenting antibody predominates for the first month and is gradually replaced by slowly sedimenting antibody.
3. The slowly sedimenting antibody activity as well as that of the rapidly sedimenting antibody activity is almost entirely destroyed by 2-mercaptoethanol treatment.
4. No anamnestic response occurred.
5. The precipitin reaction between turtle antibody and antigen remains, throughout the course of immunization, similar to a mammalian primary response in that it is greatly effected by dilution of the test system.
When human and rabbit 7S γ-globulins were reduced in strong urea solutions by a number of procedures, their molecular weights fell to approximately ⅓ of the original values. Partial separation of the reduction products was achieved using chromatography and starch gel electrophoresis in urea solutions. One of the components of reduced human 7S γ-globulin was isolated by chromatography, identified by starch gel electrophoresis, and subjected to amino acid analyses. The amino acid composition of this component differed from that of the starting material and also from that of the remaining components.
A reduced pathological macroglobulin dissociated to components with an average molecular weight of 41,000. Several reduced human myeloma proteins, when subjected to starch gel electrophoresis, yielded individual patterns that nevertheless had features in common with those of reduced normal γ-globulins. Reduction of normal and abnormal γ-globulins was accompanied by the appearance of titratable sulfhydryl groups. Chemical treatments other than reduction were used to determine the type of bond holding the subunits together. It was tentatively concluded that they were linked by disulfide bonds. An hypothesis is presented to relate the structural features of the various γ-globulins in terms of the multiplicity of polypeptide chains in these molecules.
The antigenic properties of the polypeptide chains of human 7S γ-globulin have been related to two major non-cross-reacting antigenic determinants of the whole molecule. These determinants, called S and F, were obtained by hydrolysis of γ-globulin with papain. Antisera against whole γ-globulin and against S and F fragments were used in techniques of immune diffusion. Light (L) chains of γ-globulin showed reactions of partial identity with S fragments, and thus are antigenically deficient with respect to these fragments. Antisera directed against F determinants did not react with L chains but did react with heavy (H) polypeptide chain preparations. In addition, the major component of H chain fractions did not appear to contain determinants in common with the S fragments.
L chains of a γ-myeloma protein were shown to be antigenically deficient with respect to the whole myeloma molecule, and antigenically identical with the Bence-Jones protein of the same patient.
Correlation of these results with those of previous investigations have led to the conclusions that the S fragment which is known to contain the combining region of antibody molecules, consists in part of L chains or portions of L chains, and that the F fragment, which mediates several other functions of the whole molecule, is composed in part of portions of H chains.
L polypeptide chains of myeloma globulin and Bence-Jones protein isolated from the same patient were found to be identical after comparison of their tryptic hydrolysates by two-dimensional high voltage electrophoresis. The patterns of peptides from proteins belonging to antigenic group I differed markedly from those of proteins in antigenic group II. A partially purified H chain fraction was compared with L chains from the same myeloma protein. The tryptic hydrolysates yielded dissimilar patterns of peptides.
These data indicate that γ-myeloma proteins contain two kinds of polypeptide chains, Hγ chains and either LI or LII chains. The L chains appear to be identical with those comprising the Bence-Jones protein from the same patient.
After a single injection of 108–10 bacteriophage ΦX 174, the chicken, frog and goldfish were shown to produce approximately the same levels of neutralizing, rapidly sedimenting, γ-globulin antibodies as those previously obtained in analogously immunized mammals(5,6). Repeated injections of bacteriophage in the frog and goldfish, at intervals of 2–4 weeks, did not elicit an anamnestic antibody response. However, higher levels of antibody, mainly in the slowly sedimenting γ-globulin fraction were produced after immunization with bacteriophage in complete Freund's adjuvant, and, in the case of the goldfish, after further elevation of the environmental temperature to 32°C. Thus, in 3 classes of non-mammalian vertebrates a change was observed in the sedimentation properties of antibody γ-globulins produced during immunization. This change appeared similar to the replacement of 19S by 7S antibodies in the circulation of immunized mammals. These findings suggest that the mechanisms responsible for this phenomenon were present in the most recent common ancestors of terrestrial vertebrates and bony fish and that formation of rapidly sedimenting antibody is an integral and important part of the immune mechanism.
Admixture of separated L and H polypeptide chains of 7S γ-globulins under appropriate conditions has been found to result in the reconstitution of 7S molecules. The chains were mixed in 0.5 N propionic acid and when dialyzed into neutral aqueous buffers interacted to form reconstituted material in greater than 30 per cent yield. This material had sedimentation coefficients of 6S to 7S, a weight average molecular weight of 160,000, and its antigenic structure and electrophoretic properties were the same as those of 7S γ-globulin.
By the use of I131 and I125 labels on the different types of chains, combined with ultracentrifugation of chain mixtures in sucrose density gradients, the 7S product was found to contain both isotopes in ratios consistent with the presence of two L and two H chains. After hydrolysis with papain, the reconstituted material yielded products resembling S and F fragments. All of the isotope corresponding to L chains was found in the counterpart of the S fragment; the isotope corresponding to the H chain fraction was present in both fragments. The activity reconstituted from chains of a purified guinea pig antibody to f1 phage was found to be associated mainly with the 7S material. Hybrid molecules containing rabbit L chains and human H chains and of human L chains and rabbit H chains were formed by the same techniques of reconstitution.
It was found that the interchain disulfide bonds of native 7S γ-globulins could be broken and reoxidized, as could those of reconstituted 7S material. Reduced L and H chains mixed in propionic acid, dialyzed against neutral buffers containing mercaptan, then against neutral buffers in the absence of mercaptan, formed stable 7S molecules of molecular weight 160,000, which were dissociable only after reduction.
The relationships between the polypeptide chains of gammaG immunoglobulin and fragments of the molecule produced by papain and pepsin have been investigated. Specific procedures were employed including peptide mapping of tryptic hydrolysates and analysis of molecules reconstituted from chains labeled with different iodine isotopes. By these means, the Fab fragment was shown unequivocally to consist of the light chain and a portion of the heavy chain, the Fd fragment. The Fc fragment was found to be comprised of the residual portions of the heavy chain. These findings support the gross arrangement of chains embodied in recent models of the gammaG immunoglobulin molecule. The present studies have also provided additional information on the susceptibility of gammaG immunoglobulin to proteolytic cleavage. It was found that the portion of heavy chains corresponding to the Fd fragment was extensively cleaved by papain.
The chapter discusses the structure and biological activity of immunoglobulins: the structure and linkages of the four recognized chains of immunoglobulins, the positions of their various genetic markers, and the nature of the antibody-combining sites, and the existence of two separate chains within the A or heavy chain. Biological properties of immunoglobulins such as antigenic properties, allotypes of immunoglobulins, urinary excretion of immunoglobulin fragments, transfer of antibodies from mother to fetus, fixation of antibody to skin, complement fixation, distribution and turnover of immunoglobulins, and synthesis of antibodies, chemical properties such as amino acid analysis, peptide patterns, enzymatic splitting of immunoglobulins, reduction of immunoglobulins, stability of the disulfide bonds in rabbit IgG, structural relationships of IgG, IgM, and IgA, Carbohydrate content of immunoglobulins ,possibility of three types of peptide chains,position of the antibody-combining site, and heterogeneity of immunoglobulins, physical studies such as molecular weight, electron microscope studies, and tertiary structure are also discussed in this chapter.
Treatment of sheep erythrocytes with suitable concentrations of tannic acid render them capable of adsorbing certain protein molecules from solution in saline. Red cells which have adsorbed proteins in this way are agglutinated after washing by the homologous antiprotein sera, even by high dilutions.
Through hemagglutination sera can be titrated for antibodies against antigens adsorbed on the cells exposed to tannic acid. Furthermore, small amounts of the antigens can be detected through their power to inhibit hemagglutination of the treated cells.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Dissociation of 7-globulin
- G M Edehnan
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