Immunoglobulin classes in the garden lizard,Calotesversicolor

Two classes of immunoglobulins have been purified from lizard serum using a combination of ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 or on Sepharose 6B. Lizard IgM is 2-ME sensitive and has an intact molecular weight similar to human IgM. On SDS-PAGE, reduced IgM dissociates into heavy and light chains of molecular weight 70,000 and 23,500 daltons respectively. Lizards also possess a 2-ME resistant, low molecular weight immunoglobulin designated as IgY similar to avian and amphibian IgY. IgY dissociates on SDS-PAGE to yield 59,500 dalton heavy and 26,000 dalton light chains. Antisera raised in rabbits to each of the two Ig classes could be made class-specific by cross-absorption, thus indicating that IgM and IgY represent distinct isotypes.     

Purification and partial characterization of an IgM-like serum immunoglobulin from Atlantic salmon (Salmo salar)

An IgM-like immunoglobulin was isolated from pooled sera collected from healthy Atlantic salmon. The immunoglobulin was purified by means of gel filtration followed by ion-exchange chromatography. It eluted from the ion-exchange column as two distinct peaks, but the two IgMs seems to be identical in their molecular natures except for net charge. The molecular weight of the unreduced (native) IgM was determined to be approximately 800 kilo Daltons (kD) when estimated by dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). However, when the molecular weight of the native IgM was estimated by gel filtration, a molecular weight of 1000 kD was obtained. Furthermore, the molecular weights of the heavy and the light chains were estimated by SDS-PAGE analysis to be about 72 and 27 kD respectively. The amount of IgM was found to range from 80 to 130 mg/100 ml serum. Isoelectric focusing demonstrated that the major part of the IgM molecules focused between pH 5 and pH 6.     

Purification and partial characterization of an IgM-like serum immunoglobulin from atlantic salmon ( )

An IgM-like immunoglobulin was isolated from pooled sera collected from healthy Atlantic salmon. The immunoglobulin was purified by means of gel filtration followed by ion-exchange chromatography. It eluted from the ion-exchange column as two distinct peaks, but the two IgMs seems to be identical in their molecular natures except for net charge. The molecular weight of the unreduced (native) IgM was determined to be approximately 800 kilo Daltons (kD) when estimated by dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). However, when the molecular weight of the native IgM was estimated by gel filtration, a molecular weight of 1000 kD was obtained. Furthermore, the molecular weights of the heavy and the light chains were estimated by SDS-PAGE analysis to be about 72 and 27 kD respectively. The amount of IgM was found to range from 80 to 130 mg/100 ml serum. Isoelectric focusing demonstrated that the major part of the IgM molecules focused between pH 5 and pH 6.     

Spatial structure of immunoglobulin molecules

Summary Immunoglobulin molecules of the class G (antibody molecules) consist of two heavy chains (50,000 dalton molecular weight) and two light chains (25,000 dalton). The overall shape is a Y with the arms formed by the light chains and the N-terminal half of the heavy chains in tight association. The stem is formed by the C-terminal halfs of the heavy chains.The heavy and the light chains fold into globular domains of molecular weights of 12,000 dalton. There are four domains of the heavy chain and two of the light chain. All these domains show a similar fold, consisting of two -sheets but display considerable differences in detail.The N-terminal variable domains of heavy and light chains and specifically the hypervariable polypeptide segments of the domains, located at the tips of the Y, constitute the antigen and hapten binding site. The nature of the amino acid residues of the hypervariable loops determines the shape and the specificity of the antibody.All domains pair tightly laterally, except the C_H2 domains of the heavy chain. This domain has carbohydrate bound which prevents lateral association.Longitudinal interaction between the domains is loose and allows flexibility in the arrangement. Flexibility is probably of significance for antibody function.Arm (Fab) and stem (Fc) parts are linked by the hinge peptide which contains a segment with a unique conformation of two parallel poly-proline helices.Antigen binding triggers effector functions of antibodies. Antigen binding is at the tips of the Y-shaped antibody, but effector functions are displayed by the stem part. It is an open question whether conformational changes of the antibody molecule play a significant role in the trigger mechanism.     

Identification of a second immunoglobulin in the most primitive shark, the frill shark, Chlamydoselachus anguineus.

Three molecular forms of immunoglobulins: pentamer, dimer and monomer, were isolated from serum of the frill shark, Chlamydoselachus anguineus, the most primitive extant shark. A pentamer having an apparent mass of 900 kDa, consisting of 68 kDa heavy (H) chains and 22-24 kDa light (L) chains, was considered to be IgM because of its similarity to mammalian IgM in both molecular form and H chain molecular weight. The dimer and monomer with apparent masses of 300 kDa and 150 kDa, respectively, were composed of the same H chains of 45-50 kDa, a value smaller than that of the H chains from the pentamer, and light chains identical to those of the pentamer. The H chains of 68 kDa and 45-50 kDa represented individual epitopes and were synthesized by different plasma cells. We thus concluded that the frill shark has two distinct classes of immunoglobulins: one a pentameric IgM and the other a second class of immunoglobulin with dimeric and monomeric structure. The presence of the second Ig is of considerable interest with respect to antigen recognition and exclusion.     

Expression of heavy chain‐only antibodies can support B‐cell development in light chain knockout chickens

Since the discovery of antibody‐producing B cells in chickens six decades ago, chickens have been a model for B‐cell development in gut‐associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJC_L knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL^?/?) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy‐chain‐only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4‐week‐old IgL^?/? chickens, and antigen‐specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy‐chain‐only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B‐cell development in a gut‐associated lymphoid tissue species.     

HEAVY CHAINS OF MURINE SPLENOCYTE MEMBRANE IMMUNOGLOBULINS: A COMPARISON WITH THE HEAVY CHAINS OF HUMAN AND TOAD SERUM IMMUNOGLOBULINS

The mobilities of murine splenocyte surface immunoglobulin heavy chains were compared by polyacrylamide gel electrophoresis in SDS-containing buffers, with those of the heavy chains of human IgM, IgG, and IgD, and toad IgY. Human δ-chain showed a mobility only slightly faster than that of human μ-chain, and required double-labelling techniques for unequivocal resolution. The mobility of the slower murine splenocyte surface heavy chain was identical to that of human μ-chain, but that of the second splenocyte heavy chain was significantly faster than that of human δ-chain, and similar to that of the heavy chain of toad IgY. Although the second murine splenocyte heavy chain is clearly distinct from μ-chain, no physico-chemical evidence exists which relates it directly to human δ-chain.     

Characterization of immunoglobulins from the brown bullhead (Ictalurus nebulosus) produced against a naturally occurring bacterial pathogen, Aeromonas hydrophila

The primary immune response in the brown bullhead (Ictalurus nebulosus) to intramuscularly injected, chloroform-killed Aeromonas hydrophila reached an agglutination titer of 128 in 3 wk, and the response to the bacteria plus adjuvant reached a titer of 512 in 5 wk. Anti-Aeromonas hydrophila antibodies from brown bullheads were excluded from Sephadex G-200 and had an immunoelectrophoretic migration pattern toward the cathode. Cleavage of the macromolecule by 2-ME reduction and iodoacetamide alkylation yielded subunits that were resumptively heavy chains of about 50,500 daltons and light chains of about 22,500 daltons. Bullhead antibodies were assumed to be tetrameric in configuration and therefore to have an approximate molecular weight of 600,000 daltons assuming a J chain is present in both early (6 wk) and late (20 wk) immune sera. The agglutinating activity of the antibody was completely eliminated by exposure to 65 degrees C for 30 min. Immunoelectrophoresis showed a single antibody component that moved toward the cathode. The structure and molecular weight of bullhead antibodies appears similar to that observed in channel catfish and other closely related teleost fish.     

Characterization of axolotl heavy and light immunoglobulin chains by monoclonal antibodies

Axolotl specific antibodies to 2,4-dinitrophenyl (DNP) were purified by affinity chromatography from the sera of animals immunized with 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC). The purified anti-TNP/DNP antibodies, when analyzed by SDS-PAGE, were constituted of high molecular weight molecules, which in reducing conditions, were separated into heavy 72-88 kD and light 27-30 kD polypeptides. The axolotl heavy antibody chains strongly bound Concanavalin-A and migrate faster in SDS-PAGE after endoglycosidase-F (Endo-F) treatment. Using the same techniques, no carbohydrate components were detected onto light chains. Monoclonal antibodies (MAbs) were obtained against these purified axolotl immunoglobulins (Ig) and their specificities were studied by immunoblotting. MAbs 33.45.1 and 33.101.2 respectively recognized heavy and light chains determinants of the Ig molecule. These determinants were resistant to Endo-F digestion, suggesting that the two MAbs were not directed to polypeptide-associated N-linked high mannose or complex oligosaccharides. MAbs 33.45.1 and 33.101.2 were compared to 11.5.2, an anti-axolotl thymocytes MAb which was reactive for both axolotl leucocytes and soluble Ig. MAb 11.5.2 reacted in immunoblotting against several high molecular weight axolotl serum proteins, including heavy Ig chains. Light chains were not recognized. However, 11.5.2 did not further recognize Endo-F treated Ig, suggesting its specificity for a carbohydrate determinant of the heavy chain, and link to a large diversity of soluble or membrane glycoproteins.     

Studies on Xenopus laevis immunoglobulins

The anuran amphibian Xenopus laevis has been shown to produce two classes of antibodies to HGG, BSA and Hc. These antibodies were characterized by gel filtration on Sephadex G-200 as `19S'' and `7S'' immunoglobulins. In the course of immunization, antibody activity could be initially detected in the `19S'' immunoglobulin fraction, followed by the appearance of the activity in the `7S'' immunoglobulin fraction at a later stage of immunization. A switch-over from `19S'' to `7S'' activity was not observed. Both immunoglobulins were composed of heavy and light polypeptide chains. The `19S'' protein had heavy chains with a molecular weight of 74,500, similar to human μ-chain (73,900). The `7S'' protein differed from human IgG in respect to the molecular weight of its heavy chain which was shown to be 64,500. Light chains of both immunoglobulins of Xenopus were found to have a molecular weight of 26,700, similar to human immunoglobulin light chains (25,000).     

Ontogenic studies on the appearance of two classes of immunoglobulin-forming cells in the spleen of the Aleutian skate, Bathyraja aleutica, a cartilaginous fish

We identified the presence in the Aleutian skate, Bathyraja aleutica, of two classes of immunoglobulins (Ig), a high molecular weight Ig analogous to mammalian IgM and a low molecular weight Ig, similarly to the spiny rasp skate, Raja kenojei, (Kobayashi, K. et al., Mol. Immunol. 1984. 21: 397), using an immunological cross-reaction with the specific antisera to the spiny rasp skate Ig components. The antigenic similarity of the heavy chains of the Ig of the Aleutian skate and those of the spiny rasp skate was less than that between their light chains. Two types of Ig-producing cells, one producing the high molecular weight and the other forming the low molecular weight Ig, were present in the spleen of embryos and adults in the Aleutian skate at a ratio of 5-6:4-5. The number of these Ig-producing cells increased with advancing development of the embryos but was 1/20 to 1/50 of those of adults. Cells, each of which were capable of forming both classes of Ig, were found in the spleen of embryos but not in that of adults. These results suggested that the spleen of the Aleutian skate is the primary lymphoid organ for B lymphocyte differentiation and proliferation, possibly equivalent to the bursa of birds.     

Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck

Galliform and non-galliform birds express three immunoglobulin isotypes, IgM, IgA and IgY. Beyond this we should not generalize because differences in gene organization may have functional consequences reflected in the immune response. At present, studies on non-galliform birds are largely restricted to ducks. Ducks express an alternatively spliced form of their IgY heavy chain (upsilon) gene, the IgY(DeltaFc), that lacks the Fc region and Fc-associated secondary effector functions. It is not known how common the expression of the IgY(DeltaFc) is among birds, nor the functional consequences. It is also not known whether the unusual organization of the duck IgH locus, also shared with the chicken, having the gene order of mu, alpha and upsilon, with alpha inverted in the locus, is unique to the galloanseriform lineage. Ducks, like chickens, have a single immunoglobulin light chain of the lambda (lambda) type. Evidence suggests that ducks, like chickens, generate their immunoglobulin repertoire through a single functional rearrangement of the variable (V) region, and generate diversity through gene conversion from a pool of pseudogenes. In Southern blots of germline and rearranged bursal DNA, both the heavy and light chain loci of ducks appear to each undergo one major rearrangement event. For both heavy and light chains, the functional V region element and the pseudogenes appear to consist of a single gene family. Further analysis of 26 heavy chain joining (JH) and 27 light chain JL segments shows there is use of a single J segment in ducks, which is diversified presumably through somatic mutations and gene conversion events. Despite this limitation on the rearrangement of immunoglobulin genes, analysis of 26 DH and 122 VL sequences suggests that extensive sequence diversity is generated.     

Studies on Xenopus immunoglobulins using monoclonal antibodies

Monoclonal antibodies raised against Xenopus Ig recognize antigenic determinants on IgM and low mol. wt Ig (LMW Ig or IgY ). On SDS-PAGE two forms of mu were distinguished in the supernatant and cell lysate of tunicamycin-treated spleen cell culture. The two bands of v observed in serum appear to be the result of carbohydrate heterogeneity. The light chains resolved into two distinct bands which differed in peptide maps; the more slowly migrating band of light chain was preferentially associated with v. Patterns of cross-reactivity between the immunoglobulins of 11 species of Xenopus and three subspecies of Xenopus laevis were obtained; the polymorphism of mu and v antigenic determinants and its implications for species divergence are discussed.     

Major immunoglobulin classes of the Echidna (Tachyglossus aculeatus)

The Australian echidna responds to the antigen Salmonella adelaide flagella by producing antibodies characterized by mol. wt of 900,000 and 150,000. After cleavage of interchain disulphide bonds, both the high and low mol. wt immunoglobulins can be resolved into light and heavy polypeptide chains. In both cases, the light chains resemble those of other vertebrate immunoglobulins in size (22,500 Daltons) and electrophoretic mobility. The 900,000 Dalton immunoglobulin contains heavy chains similar to human μ chains in size (70,000 Daltons) and electrophoretic mobility. The 150,000 Dalton immunoglobulin contains a different class of heavy chain, similar in size (50,000 Daltons) and electrophoretic mobility to human γ chains. Proportional mass contributions of the light and heavy chains to the intact molecule suggest the structure of the intact molecules could be represented by (L₂, μ₂)₅ and (L₂, γ₂) for the high and low mol. wt immunoglobulins respectively. These configurations are similar to those described for human γM and γG immunoglobulins.The results are relevant to theories of the evolution of the different classes of immunoglobulins. While the echidna is distinctly more primitive than eutherian mammals and still retains structural features characteristic of reptiles, its major immunoglobulin classes are very similar to human IgM and IgG. The striking similarities between the γ-like heavy chain of the echnidna and human IgG heavy chains suggest that the echidna may be the first species in which a γ chain gene directly homologous to mammalian γ chain genes is expressed.     

Expression of surface and secreted IgG22a by a murine B-lymphoma before and after hybridization to myeloma cells

A20 is a spontaneous Balb/c murine lymphoma which has been reported to express immunoglobulin, Fc receptors, and la antigens, but not complement receptors, on its cell surface. Approximately 0.1% of the protein synthesized by a subclone of A20 (A20-1.11) is IgG_2a,which is present on the cell surface as well as secreted by the cells. Biosynthetic labeling studies reveal that A20-1.11 cells synthesize a single κ light chain as well as two species of intracellular γ_2a heavy chain. The smaller γ_2a heavy chain, with a molecular weight of 62,000 daltons, is the intracellular precursor to a 64,000 dalton γ_2a heavy chain which is present in secreted IgG_2a. The larger intracellular γ_2a heavy chain, with a molecular weight of 68,000 daltons, is the intracellular precursor to a 70,000 dalton γ_2a heavy chain which is present in surface IgG_2a. Fusion of A20-1.11 lymphoma cells to murine myeloma cells results in hybrids expressing the more differentiated myeloma phenotype. That is, the expression of the secreted form of the lymphoma IgG_2a is amplified as much as 150 fold while the expression of the surface form of the lymphoma IgG_2a is abolished in the hybrid. The relationship of the γ_2a heavy chains expressed in these cells is discussed in relation to what has been reported for surface and secreted μ heavy chains.     

Immunoglobulins of the Australian brush-tailed opossum, Trichosurus vulpecula

Using standard methods IgG, IgM and (s) IgA were isolated from the serum or intestinal fluid of the Australian brush-tailed opossum, Trichosurus vulpecula. The molecular weights of the intact immunoglobulin molecules and their heavy and light chains were established. Two forms of IgG were seen which differed in their abilities to bind to insoluble matrices and also in their molecular weights. No antigenic differences were seen between them on analysis by agar diffusion. The molecular weights of opossum IgM and the two forms of IgG were similar to those of the corresponding immunoglobulins of man and other marsupials. However, the molecular weight of the IgA seen in intestinal fluid and results from its analysis by agar diffusion suggest that the molecule may lack secretory component.     

The structure of two IgMs showing different activity from a patient with Waldenstrom''s macroglobulinaemia.

The two monoclonal IgMs (IgM1 and IgM2) were characterized from a patient Waldenström''s macroglobulinaemia that resulted in a gammapathy. Heavy and light chains were isolated from the IgM. The complete primary structure of the two light chains and the NH2-terminal region of the two heavy chain molecules were determined. The sequence data indicated that the heavy and light chains from both IgMs belong to the same (III and II) lambda subgroups. By testing their antibody activity it was found by immunofluorescence and immunoblotting that only IgM2 reacts with an intermediate filament protein.     

Monoclonal antibodies to axolotl immunoglobulins specific for different heavy chains isotypes expressed by independent lymphocyte subpopulations

An immunoblotting analysis of purified axolotl immunoglobulins (Ig) separated by SDS-PAGE reveals two heavy (H) chains isotypes: a 76 kDA chain recognized by the monoclonal antibody (mAb) 33.45.1 and a 66-68 kDa doublet recognized by the mAb 33.39.2. The 76 kDa chain is associated to high molecular weight (HMW) Ig molecules and the 66-68 kDa H chains are associated to low molecular weight (LMW) Ig of 172 kDa. Both H chains isotypes are linked to identical light (L) chains, labelled in immunoblotting by the mAb 33.101.2. Two different axolotl lymphocyte subpopulations are characterized by these two distinct H chains isotypes. One population of splenic lymphocytes (approximately 40%) is labeled by indirect immunofluorescence with mAb 33.45.1, specific for the 76 kDa H chain isotype. Another population (approximately 20%) is labeled by mAb 33.39.2 specific for the 66-68 kDa H chain isotype. Both populations of splenic lymphocytes are stained by mAb 33.101.2 specific for the axolotl L chains. Therefore, the presence of at least two independent Ig classes is now confirmed in a urodele amphibian species at the humoral and cellular levels.     

A novel interaction involving a polypeptide chain (P33) in covalent linkage with IgM on the surface of a Burkitt lymphoma cell line (Daudi).

The surface IgM of Daudi Burkitt lymphoma cells has been shown to consist of mu chains and kappa chains covalently linked, by disulfide bonding, to a novel polypeptide chain of approximate molecular weight 33,000. This novel polypeptide chain (P33) is similar in several properties to the heavy chain of the Ia antigen: glycoprotein nature; apparent molecular weight; isoelectric behavior and cysteine content. The linkage of P33 to surface immunoglobulin has not been observed with any other human lymphoma or lymphoblastoid cell lines.