IgX antibodies in the urodele amphibian Ambystoma mexicanum

Until recently, it was believed that urodele amphibians are able to synthesize only two immunoglobulin isotypes, IgM and IgY. We reinvestigated this issue in the Iberian ribbed newt Pleurodeles waltl and reported recently that this urodele expresses at least three isotypes: IgM, IgP and IgY. In this study, we demonstrate that another urodele, Ambystoma mexicanum, has also a third isotype whose amino acid sequence presents the highest homology with the amino acid sequence of Xenopus IgX. This isotype has typical Ig H-chain characteristics, could form multimers and is mainly expressed in mucosal tissues thereby indicating that it is likely the physiological counterpart of Xenopus IgX and mammalian IgA. Interestingly, no IgP could be found in A. mexicanum, in contrast to P. waltl, in which IgX was not found in previous investigations. These data indicate, for the first time, that different families of urodeles can express different immunoglobulin isotypes.     

Is Xenopus IgX an analog of IgA?

Using class specific monoclonal antibodies we analyzed the tissue distribution of B cells expressing the three immunoglobulin (Ig) isotypes (IgM, IgX, IgY) in Xenopus. Large numbers of IgM- and IgX-, but not IgY-, positive B cells are located in the gut epithelium of the intestine. In this organ up to 60% of all B cells can be IgX positive, while in the spleen or liver they are hardly detectable. The majority of IgX-producing cells resemble plasma cells. IgY-producing cells are found in the liver and spleen but not in the intestine. In contrast to IgY, the expression of IgM and IgX is thymus independent. Upon systemic immunization, a several-fold increase of specific IgM and IgY, but not IgX, antibodies was detected in the sera. This and its association with the mucosae of the intestine resembles results reported for mammalian IgA; therefore, IgX of Xenopus might be considered an analog of IgA.     

Cloning and sequencing of the immunoglobulin A1 protease gene (iga) of Haemophilus influenzae serotype b.

Secretion of immunoglobulin A1 (IgA1) proteases is a characteristic of Haemophilus influenzae and several other bacterial pathogens causing infectious diseases, including meningitis. Indirect evidence suggests that the proteases are important virulence factors. In this study, we cloned the iga gene encoding immunoglobulin A1 (IgA1) protease from H. influenzae serotype b into Escherichia coli, in which the recombinant H. influenzae iga gene was expressed and the resulting protease was secreted. Sequencing a part of a 7.5-kilobase DNA fragment containing the iga gene revealed a large open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,541 amino acids and a molecular mass of 169 kilodaltons. Putative promoter and terminator elements flanking the open reading frame were identified. Comparison of the deduced amino acid sequence of this H. influenzae IgA1 protease with that of a similar protease from Neisseria gonorrhoeae revealed several domains with a high degree of homology. Analogous to mechanisms known from the N. gonorrhoeae IgA protease secretion, we propose a scheme of posttranslational modifications of the H. influenzae IgA1 protease precursor, leading to a secreted protease with a molecular mass of 108 kilodaltons, which is close to the 100 kilodaltons reported for the mature IgA1 protease.     

Immunoglobulin evolution: chemical study of clawed toad (Xenopus laevis) heavy and light chains.

NH2 terminal amino acid sequence determinations of clawed toad (Xenopus laevis) immunoglobulins indicate that approximately 30% of the heavy chains and less than 5% of the light chains have unblocked NH2 termini. The major amino acid sequence of the X. laevis 7S immunoglobulin heavy chains is the same as that of the 19S immunoglobulin heavy chains. Thus in the synthesis of the heavy chains, the VH genes coding for unblocked heavy chains can associate with CH genes of both the 19S and 7S classes. This association is particularly important in amphibians because, in contrast to mammals and birds, the majority of amphibian antibody-producing cells synthesize both 19S and 7S immunoglobulins and do not participate in the 'genetic switch' characteristic of lymphocyte differentiation in higher organisms. In X. laevis, the major amino acid sequence at the first twenty-four positions of the unblocked heavy chains shows approximately 54% difference from the prototype amino acid sequence of the mammalian VHIII subgroup. Thus, the VHIII gene(s) must have started to appear after the evolutionary divergence of the common ancestor of mammals and birds from the amphibian line. The amino acid composition of the X. Laevis 7S immunoglobulin heavy chains differs from that of its 19S immunoglobulins as well as those of human IgG and IgA. These data support the concepts (a) that amphibian 7S and 19S immunoglobins belong to distinct classes and (b) that amphibian 7S immunoglobulin does not resemble mammalian IgG or IgA.     

Humoral response of chicken infected with the microsporidium Encephalitozoon hellem

Chicken (Gallus gallus) were used as the experimental model for study of immune response against the microsporidium Encephalitozoon hellem (Didier et al., J Inf Dis 163:617–621, 1991) infection in birds. Two-day-old chicken were infected perorally or intraperitoneally with a dose of 10⁷ spores of E. hellem. The anti-E. hellem immunoglobulin (Ig)A, IgY, and IgM antibody responses in sera and dropping sample extracts were determined by enzyme-linked immunosorbent assay. Results have shown specific antibody production in sera and intestinal secretions of infected birds. Chicken inoculated perorally developed the lowest antibody response. Microsporidian spores were not identified in the smears from cloacal swab samples of individual chicken. Intestinal segment cultures of perorally infected chicken cultivated in vitro showed the highest production of specific IgY and IgA antibodies in jejunum segments. In the further course of infection, the colon produced the highest amount of IgA, and the ileum and colon produced the highest amount of IgY.     

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.     

Evidence for an early appearance of modern post-switch isotypes in mammalian evolution; cloning of IgE,IgG and IgA from the marsupial Monodelphis domestica

In birds, reptiles and amphibians the IgY isotype exhibits the functional characteristics of both of IgG and IgE. Hence, the gene for IgY most likely duplicated some time during early mammalian evolution and formed the ancestor of present day IgG and IgE. To address the question of when IgY duplicated and formed two functionally distinct isotypes, and to study when IgG and IgA lost their second constant domains, we have examined the Ig expression in a non-placental mammal, the marsupial Monodelphis domestica (grey short-tailed opossum). Screening of an opossum spleen cDNA library revealed the presence of all three isotypes in marsupials. cDNA clones encoding the entire constant regions of opossum IgE (ϵ chain), IgG (γ chain) and IgA (α chain) were isolated, and their nucleotide sequences were determined. A comparative analysis of the amino acid sequences for IgY, IgA, IgE and IgG from various animal species showed that opossum IgE, IgG and IgA on the phylogenetic tree form branches clearly separated from their eutherian counterparts. However, they still conform to the general structure found in eutherian IgE, IgG and IgA. Our findings indicate that all the major evolutionary changes in the Ig isotype repertoire, and in basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the evolutionary separation of marsupials and placental mammals.     

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.     

Immunoglobulin Fc receptor molecules on Xenopus laevis splenocytes.

The existence of membrane-associated Fc receptors for IgY (Fc nu R) or IgM (Fc mu R) was demonstrated on a large percentage of Xenopus splenocytes. The Fc receptors were detected by direct fluorescent staining in which the spleen cells were incubated with fluorescein isothiocyanate (FITC)-conjugated antigen-complexed IgY antibodies or with FITC-conjugated heat-aggregated IgM. Results showed that 28.9% (SD +/- 5.1) and 5.3% (SD +/- 2.2) of the cells bear Fcnu or Fc mu receptors, respectively. The specificity of the receptors was tested after incubation of the cells in the presence of the following fluorochrome-conjugated reagents: non-aggregated Xenopus Igs and human IgG, aggregated Xenopus albumin, Fc6 nu and Fab mu fragments and human IgG, and antigen-complex Fab2 nu fragments. Results indicated that the receptors are specific for Xenopus immunoglobulins, with the restriction that the latter must be presented in a complexed or aggregated form to the cells and that the precise binding site is located on the Fc portion of IgY or IgM. The identity of a large percentage (22.4 +/- 2.8%) of cells bearing Fc nu R was established by direct simultaneous double-fluorescent staining of surface membrane IgM and Fc nu R. All Xenopus splenic B lymphocytes bearing sIgM carry also Fc nu R, while 8.8% of splenocytes, of yet unknown lineage, bear Fc nu R alone on their surface. The presence of Fc receptors on Xenopus B lymphocytes (leaving aside their eventual presence in other leucocyte types) suggests, once again, a high degree of evolution of the immune system in these lower vertebrates.     

The Origin of the Immune System

It has been suggested that the neural cell adhesion molecules (N-CAM) are members of the Ig superfamily. We have examined structural homology between the N-CAM and other superfamily molecules in detail. The Ig-like domains of N-CAM have overall sizes similar to those of Ig constant domains or major histocompatibility complex (MHC) domains. Significant sequence homology was found between the N-CAM Ig-like domains and several other Ig superfamily members, such as T4, T8, IgA transport receptor, Ig VH, Ig C mu 4, and others. A comparison of the second domain of T4 (255 amino acids) with the central domain of N-CAM (302 amino acids) has demonstrated a significant sequence homology. Furthermore, a cell adhesion protein, cs-A, of slime mould, Dictyostelium, also seems to be related to N-CAM. It appears that the Ig superfamily of the immune system originated from more ubiquitous cell recognition molecules which are common in all metazoan species.     

Analysis of the immunoglobulin A protease gene of Streptococcus sanguis.

The amino acid sequence T-P-P-T-P-S-P-S is tandemly duplicated in the heavy chain of human immunoglobulin A1 (IgA1), the major antibody in secretions. The bacterial pathogen Streptococcus sanguis, a precursor to dental caries and a cause of bacterial endocarditis, yields IgA protease that cleaves only the Pro-Thr peptide bond in the left duplication, while the type 2 IgA proteases of the genital pathogen Neisseria gonorrhoeae and the respiratory pathogen Haemophilus influenzae cleave only the P-T bond in the right half. We have sequenced the entire S. sanguis iga gene cloned into Escherichia coli. A segment consisting of 20 amino acids tandemly repeated 10 times, of unknown function, occurs near the amino-terminal end of the enzyme encoded in E. coli. Identification of a predicted zinc-binding region in the S. sanguis enzyme and the demonstration that mutations in this region result in production of a catalytically inactive protein support the idea that the enzyme is a metalloprotease. The N. gonorrhoeae and H. influenzae enzymes were earlier shown to be serine-type proteases, while the Bacteroides melaninogenicus IgA protease was shown to be a cysteine-type enzyme. The streptococcal IgA protease amino acid sequence has no significant homology with either of the two previously determined IgA protease sequences, that of type 2 N. gonorrhoeae and type 1 H. influenzae. The differences in both structure and mechanism among these functionally analogous enzymes underscore their role in the infectious process and offer some prospect of therapeutic intervention.     

Nucleotide sequence of bovine herpesvirus type 1 glycoprotein gIII, a structural model for gIII as a new member of the immunoglobulin superfamily, and implications for the homologous glycoproteins of other herpesviruses

The gene encoding bovine herpesvirus type 1 (BHV-1) glycoprotein gIII was mapped, cloned, and sequenced. The gene is situated between map units 0.122 and 0.135 and encodes a predicted protein of 521 amino acids. The identity of the sequenced gene has been verified previously by expression of immunologically authentic recombinant BHV-1 gIII (Fitzpatrick et al. (1988) J. Virol. 62, 4239-4248). Comparison of the BHV-1 gIII amino acid sequence with the homologous glycoproteins of other alphaherpesviruses revealed significant homology in the carboxy-terminal half of the molecules, including six invariant cysteine residues. A 96 amino acid domain with significant homology to class II major histocompatibility complex (MHC) antigen constant domains was identified in the conserved carboxy-terminal half of BHV-1 gIII. This domain is flanked by two other similarly sized domains which may be related to other immunoglobulin (Ig) superfamily domains. These homologies support a model for the structure of BHV-1 gIII as a new member of the Ig superfamily. Elements of the model may be applicable to the homologous glycoproteins of other herpesviruses and relevant to the immunobiology of herpesvirus infections.     

Bile immunoglobulin of the duck (Anas platyrhynchos). II. Antibody response in influenza A virus infections

The capacity of the IgM-like bile immunoglobulin (IgX) of the duck (Anas platyrhynchos) to express antibody activity to H3N2 influenza A viruses, and the dependence of this activity on the co-existence of serum IgM antibodies were investigated. Ducklings infected orally and intranasally at 15-29 days of age with viruses isolated from different host species were examined for haemagglutination-inhibiting (HI) antibodies in biles and sera 16-29 days after infection (p.i.). All biles had antibodies associated with IgX; all sera had antibodies associated only with the 7.8S IgG. Following oral infection of birds 42-days-old with influenza A/duck/HK/7/75 virus, serum HI antibodies were an initial IgM response occurring from 5-12 days p.i., followed by the appearance of 7.8S IgG antibodies. Virus-neutralizing (VN) antibodies in serum were also biphasic; isotype classification was not attempted. Bile IgX developed HI and VN activity. HI antibodies reached peak titres 12 days p.i. and fell to low levels by 24 days p.i. VN antibodies also reached peak titres 12 days p.i., but thereafter persisted at quite high levels throughout the experiment. Development of high titres of antibody in bile coincided with the termination of virus excretion in faeces. These experiments confirm that bile IgX of the duck can function as antibody in response to influenza A viruses, and that its activity appears to be independent of serum IgM. Its possible relevance in determining survival of virus in the intestine is discussed.