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M F Flajnik

Publications and source records attributed to M F Flajnik.

At least 37 records · Page 2Linked to original sources
PubMed1998

Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: possible role in antigen-driven reactions in the absence of germinal centers.

The new antigen receptor (NAR) gene in the nurse shark diversifies extensively by somatic hypermutation. It is not known, however, whether NAR somatic hypermutation generates the primary repertoire (like in the sheep) or rather is used in antigen-driven immune responses. To address this issue, the sequences of NAR transmembrane (Tm) and secretory (Sec) forms, presumed to represent the primary and secondary repertoires, respectively, were examined from the peripheral blood lymphocytes of three adult nurse sharks. More than 40% of the Sec clones but fewer than 11% of Tm clones contained five mutations or more. Furthermore, more than 75% of the Tm clones had few or no mutations. Mutations in the Sec clones occurred mostly in the complementarity-determining regions (CDR) with a significant bias toward replacement substitutions in CDR1; in Tm clones there was no significant bias toward replacements and only a low level of targeting to the CDRs. Unlike the Tm clones where the replacement mutational pattern was similar to that seen for synonymous changes, Sec replacements displayed a distinct pattern of mutations. The types of mutations in NAR were similar to those found in mouse Ig genes rather than to the unusual pattern reported for shark and Xenopus Ig. Finally, an oligoclonal family of Sec clones revealed a striking trend toward acquisition of glutamic/aspartic acid, suggesting some degree of selection. These data strongly suggest that hypermutation of NAR does not generate the repertoire, but instead is involved in antigen-driven immune responses.

Amino Acid Sequence↗
PubMed1998

Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins.

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (VH) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.

Amino Acid Sequence↗
PubMed1998

Expression of MHC class Ia and class Ib during ontogeny: high expression in epithelia and coregulation of class Ia and lmp7 genes.

The amphibian Xenopus permits the examination of immune responses in a species that progresses through two distinct lives, tadpole and adult, in which animals are free-living and immunocompetent. MHC gene expression as well as general features of the immune system change profoundly at metamorphosis. In this study gene expression of class Ia, class Ib, and the immune proteasome component lmp7 was investigated by Northern blotting at all stages of development. Class Ia genes are expressed in most adult tissues, with highest levels in intestine. Class Ib genes are expressed at lower levels, and their tissue distribution is somewhat more restricted than that of class Ia. Consistent with the idea that particular class Ib isotypes can perform distinct functions, preferential expression of class Ib genes is found in some tissues, with one family being expressed exclusively in epithelia. The onset of MHC expression is not simultaneous in all tissues: class Ia transcripts are first present in tadpole lung, gill, and intestine, organs with epithelial surfaces in contact with the environment. In every tissue except colon and muscle, class Ia expression increases markedly after metamorphosis. Interestingly, expression of the MHC-linked proteasome component lmp7 mirrored class Ia expression, while the constitutive lmp7 homologue X was expressed ubiquitously at all stages. Class Ib transcripts were never detected before metamorphosis, suggesting that the Xenopus class Ib proteins identified to date do not subserve class Ia functions in tadpole life.

Animals↗
PubMed1998

Evolution of somatic hypermutation and gene conversion in adaptive immunity.

Examples of somatic hypermutation of antigen receptor genes can be seen in most lineages of vertebrates, including the cartilaginous fish. Analysis of the phylogenetic data reveals that two distinctive features of the mechanism are shared by most species studied: the mutation hot spot sequence AGY, and a preponderance of point mutations. These data suggest that some of the components of the machinery are shared between ectotherms and mammals. However, unique characters in particular species may have occurred by independent recruitment of novel factors onto the mechanism. A spotty phylogenetic distribution of gene conversion has also been revealed and can be explained if the two mechanisms share some characteristics. Both mutation and conversion require transcription-related sequences and/or factors. We theorized that targeting to V genes can be attained by a paused replication fork that has collided with a transcription complex stalled by a defective Ig transcription activator; the paused replication fork results in recruitment of an error-prone translesion synthesis DNA polymerase (somatic hypermutation) or of DNA repair mechanisms with homologous recombination (gene conversion). In addition, the pathway recruited in different species may be directed by the degree of homology among V genes.

Animals↗
PubMed1997

Identification of class I genes in cartilaginous fish, the most ancient group of vertebrates displaying an adaptive immune response.

Sharks are members of the most primitive class of vertebrates (Chondrichthyes) shown to have an adaptive immune system. Suprisingly, however, class I genes have not been identified unambiguously in this taxon, and absence of class I loci or a failure to express class I genes might explain some of the relatively "weak" adaptive immune responses documented in cartilaginous fish. We report here the isolation of three unique cDNA clones from two different species of sharks that encode bona fide class I proteins. These clones exhibit different sequence and expression profiles indicating that they are likely to represent both classical and nonclassical class I lineages. In addition, our preliminary analysis suggests that there may be transfer of gene segments among shark class I genes over evolutionary time. The cloning of shark class I genes completes the identification of molecules that define the adaptive immune system (including Ig, TCR, and MHC class II proteins) in this taxon. Thus, simple models invoking a total absence of certain molecular hallmarks of the immune system to account for poor immune responsiveness in cartilaginous fish should be abandoned.

Amino Acid Sequence↗
PubMed1997

Evolution of proteasome subunits delta and LMP2: complementary DNA cloning and linkage analysis with MHC in lower vertebrates.

The class II region of the mammalian MHC harbors two proteasome subunit genes, LMP2 and LMP7. These genes are induced by IFN-gamma, and their products are incorporated into proteasomes substituting for their closest relatives, the delta and X subunits, respectively. This substitution is believed to change the proteolytic specificity of proteasomes, making it more suitable for generation of peptides to be presented by class I molecules. To elucidate the phylogenetic origin of LMP2 and the linkage of its gene with the MHC, reverse transcriptase-PCR amplification of Xenopus laevis and lamprey liver mRNA was performed with primers designed to amplify both the mammalian LMP2 and delta sequences. Both LMP2 and delta were amplified from X. laevis, whereas only delta was amplified from lamprey, suggesting that delta/LMP2 gene duplication occurred after divergence of cyclostomes but before divergence of amphibians. The linkage between the LMP2 gene and the MHC was observed in a diploid Xenopus species, Xenopus tropicalis, but not in a tetraploid species, X. laevis, indicating that this linkage was established before the divergence of amphibian from higher vertebrates, but that this linkage was lost in X. laevis, probably by a gene reorganization accompanying the tetraploidization. The X. laevis LMP2 and LMP7 mRNA showed a similar tissue distribution, indicating that the genetic linkage is not required for apparently coordinated tissue-specific expression of these genes. Sequence and linkage analyses suggest that LMP2 may not play as vital a role as LMP7 in Ag presentation.

Amino Acid Sequence↗
PubMed1997

Major histocompatibility complex gene mapping in the amphibian Xenopus implies a primordial organization.

One of the most provocative recent discoveries in immunology was the description of a genetic linkage in the major histocompatibility complex (MHC) between structurally unrelated genes whose products are involved in processing and presentation of antigens for recognition by T lymphocytes. Genes encoding MHC class I molecules, which bind and present at the cell surface proteolytic fragments of cytosolic proteins, are linked to nonhomologous genes whose products are involved in the production and subsequent transfer of such fragments into the endoplasmic reticulum. In mammals, the class I presentation and processing genes are found in different regions of the MHC. To examine the evolutionary origins of this genetic association, linkage studies were carried out with Xenopus, an amphibian last sharing an ancestor with mammals over 350 million years ago. In contrast to mammals, the single copy Xenopus class I gene is located between the class II and III regions, speculated to be in close linkage with the processing and transport genes. In addition to suggesting a primordial organization of genes involved in class I antigen presentation, these linkage studies further provide insight into the origins of the MHC class III region and the phenomenon of class I gene instability in the mammalian MHC.

Alleles↗
PubMed1997

IgM-mediated opsonization and cytotoxicity in the shark.

Two types of cytotoxic reactions have been observed using cells from the nurse shark: spontaneous cytotoxicity mediated by cells of the macrophage lineage and antibody-dependent killing carried out by a different effector cell population. Previous data showed that removal of phagocytic cells using iron particles abolished macrophage-mediated killing, but not antibody-dependent reactions. The current study used single cell assays and showed that the effector of antibody-driven reactions was the neutrophil. Surprisingly, the mechanism of killing was shown to be phagocytosis mediated by both 7S and 19S immunoglobulin M (IgM). Reactions proceeded with as little as 0.01 microg of purified 19S or 7S IgM and were complete within 4-6 h. In contrast, purified immunoglobulin did not adsorb to macrophages and had no effect on target cell binding or cytotoxicity. Pretreatment of cells with cytochalasin D abolished the phagocytic reaction, but not spontaneous cytotoxicity. These data show that antibody-mediated killing results from opsonization and phagocytosis; the mechanism of macrophage killing is currently unknown. In addition, these data show that the shark neutrophil, not the macrophage lineage, carries a receptor for Fc mu.

Animals↗
PubMed1997

Involvement of thyroid hormones in the expression of MHC class I antigens during ontogeny in Xenopus.

The major histocompatibility complex (MHC) is a cluster of genes encoding products central to all major functions of the vertebrate immune system. Evidence for an MHC can be found in all vertebrate groups that have been examined except the jawless fishes. Expression of MHC class I and class II antigens early in ontogeny is critically important for development of T lymphocytes capable of discriminating self from nonself. Because of this essential role in T-cell development, the ontogeny of MHC expression in the South African clawed frog, Xenopus laevis, was studied. Previous studies of MHC class I expression in Xenopus laevis suggested that class I antigens are virtually absent from tadpole tissues until climax of metamorphosis. We therefore examined the possible role of thyroid hormones (TH) in the induction of class I. By flow cytometry, a small amount of class I expression was detectable on splenocytes and erythrocytes in untreated frogs at prometamorphic stages 55-58, and the amount increased significantly at the conclusion of metamorphic climax. Thus, metamorphosis is associated with increased intensity of class I expression. Neither inhibition nor acceleration of metamorphosis altered the timing of onset of class I expression. However, inhibition of metamorphosis prevented the increase in class I expression characteristic of adult cell populations. Because expression was not accelerated in TH-treated frogs or delayed in metamorphosis-inhibited frogs, it is unlikely that TH are the direct developmental cues that induce expression, although they seem to be required for the upregulation of class I expression occurring at metamorphosis. Differences in the pattern of expression in different sub-populations of cells suggest a complex pattern of regulation of expression of class I antigens during ontogeny.

Animals↗
PubMed1996

Isolation of low molecular mass polypeptide complementary DNA clones from primitive vertebrates. Implications for the origin of MHC class I-restricted antigen presentation.

Proteasomes are the multisubunit proteases thought to be involved in the generation of peptides presented by MHC class I molecules. When cells are stimulated with IFN-gamma, two MHC encoded subunits, LMP2 and LMP7, are incorporated into the proteasomal complex, presumably by displacing the housekeeping subunits, designated Y and X, respectively. These changes in the subunit composition appear to facilitate class I-mediated Ag presentation, presumably bu altering the cleavage specificities of the proteasome. Here we show that the cartilaginous fish, the most primitive class of vertebrates in which the MHC has been identified, have both LMP7 and X genes. Interestingly, nurse sharks, a member of the cartilaginous fish, appear to have two LMP7 genes, one encoding a typical LMP7 subunit and the other encoding a less typical one. In contrast, only cDNA clones with residues characteristic of X were identified in hagfishes and lampreys, the two extant members of the jawless fish in which no MHC has been identified. Pairwise amino acid sequence comparison and phylogenetic tree analysis showed that the subunits encoded by these clones were nearly equidistant from LMP7 and X, suggesting that the LMP7 gene might have emerged after the appearance of the jawless fish. Sequence comparison of the LMP7 and X/X-like subunits isolated from various vertebrate species showed that, unlike the X/X-like subunit, the LMP7 subunit displays a striking interspecies sequence variability in the vicinity of its catalytic site.

Amino Acid Sequence↗
PubMed1996

A novel "chimeric" antibody class in cartilaginous fish: IgM may not be the primordial immunoglobulin.

Using a degenerate oligonucleotide primer specific for immunoglobulin (Ig) constant type 1 (C-1 set) domain genes, products were amplified by the reverse transcriptase-polymerase chain reaction from nurse shark spleen cDNA. The deduced protein sequence of one of these clones reveals a novel Ig class in cartilaginous fish. A complete mRNA could encode a mature protein bearing an amino-terminal variable (V) domain, followed by six C-1 set domains, and ending in a carboxy-terminal tail typical of secreted IgM, IgA, and the new antigen receptor (NAR). The two amino-terminal C domains are orthologous to IgX (or IgR), an Ig heavy (H) chain class in the skate, and the last four domains are homologous to the carboxy-terminal four domains of NAR. We designate this "chimeric" Ig class IgNARC for Ig new antigen receptor from cartilaginous fish. Like NAR, but unlike shark IgM, IgNARC is encoded by very few V and C genes which apparently are not closely linked. The number of bands that hybridize with exon-specific probes varies with genomic DNA from individual sharks, suggestive of different numbers of IgNARC genes in different animals. A protein of approximately 95 kDa, which is likely to be the IgNARC H chain, is immunoprecipitated with both light chain-specific monoclonal antibodies and with antisera generated to a peptide comprising the IgNARC carboxy-terminal tail. We conclude that the arsenal of secreted antigen receptors in cartilaginous fish is greater than previously believed. In addition, our data cast doubt on the dogma that IgM is the primordial Ig isotype.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of a cDNA encoding a Xenopus 70-kDa heat shock cognate protein, Hsc70.I.

We isolated a full-length cDNA clone encoding a Xenopus laevis 70-kDa heat shock cognate protein, hsc70.I. The protein coding region exhibits a high degree of identity with a number of mammalian hsc70 proteins, such as rat hsc71 (92%), whereas the identity to Xenopus hsp70 is only 80%. These data suggest that the inducible and constitutive forms of hsp70 diverged long before the emergence of amphibians. The Xenopus hsc70.I contains a number of conserved elements, including the ATP-binding domain, a nuclear localization signal and the carboxy-terminal EEVD motif, which has been implicated in several activities associated with chaperonin function. Northern blot analyses revealed that maternal hsc70.I mRNA is present in cleavage and early blastula stages of Xenopus development. After the onset of zygotic transcription at the midblastula stage, the levels of hsc70.I message increase through to the tadpole stages. Furthermore, in contrast to hsp70 mRNA, the relative levels of hsc70.I mRNA are not enhanced after heat shock in embryos and in the kidney epithelial cell line, A6. The levels of hsc70.I mRNA are high in adult spleen and testis, with moderate levels in eye, heart, liver and brain and comparatively low levels in hindlimb muscle.

Amino Acid Sequence↗
PubMed1996

The immune system of ectothermic vertebrates.

The adaptive immune system, as defined by T cell receptors, immunoglobulins, and the major histocompatibility complex (MHC), has been described definitively at the level of teleost fish. Cartilaginous fish, which display many of the hallmarks of such an adaptive system, nevertheless have several features of their responses that seem primitive. Data are presented suggesting that some adaptive mechanisms in cartilaginous fish, including MHC restriction and somatic diversification, are present to the same "degree' as compared to mammals, and that these animals may possess other molecules and functions previously overlooked. MHC linkage studies in amphibians suggest that the entire genetic complex, including class I, class II, and class III genes, arose early in the vertebrate line (at least 350 x 10(6) years ago) and has been maintained intact, at least for those genes involved in immunity. Studies of MHC in polyploid Xenopus have demonstrated that there is a maximal number of expressed MHC genes 'permitted' to be expressed in any individual, regardless of the number of potential MHC-bearing chromosomes present in the species. A speculative hypothesis is presented on the origins of adaptive immunity based on ectothermic models.

Animals↗
PubMed1995

Isolation of Xenopus LMP-7 homologues. Striking allelic diversity and linkage to MHC.

The mammalian low molecular mass protein-7 (LMP-7) gene resides in the class II region of the MHC, and its product is most probably involved, as a component of a proteasome, in the processing of Ags to be presented by the MHC class I molecules. To elucidate the evolution of the LMP-7 gene at both the primary structure and genetic levels, we isolated LMP-7 cDNA clones from amphibian Xenopus laevis, which last shared a common ancestor with mammals 350 x 10(6) years ago. Two distinctive clones, showing an 85% predicted amino acid sequence identity with each other and 69 to 72% identity with human and mouse LMP-7, were identified from a liver cDNA library of outbred frogs and named XeLMP-7A and XeLMP-7B. XeLMP-7A- and XeLMP-7B-specific probes were used to detect the corresponding genes by using partially inbred frogs with known MHC haplotypes. DNA of the g and j haplotypes hybridized with the XeLMP-7A probe, whereas the f and r haplotype DNA hybridized with the XeLMP-7B probe. These hybridization patterns cosegregated with the MHC haplotypes among offspring of an f/f x f/g cross, and one recombinant revealed that the LMP-7 gene is linked more closely to class II than to class I or class III genes. Taken together, the data indicate that XeLMP-7A and XeLMP-7B are highly diverse alleles at a single locus in the frog MHC. The great allelic diversity can be explained either by coselection with particular class I alleles or by differential silencing of MHC genes in the polyploid X. laevis.

Alleles↗
PubMed1995

A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks.

Immunoglobulin and T-cell receptor (TCR) molecules are central to the adaptive immune system. Sequence conservation, similarities in domain structure, and usage of similar recombination signal sequences and recombination machinery indicate that there was probably a time during evolution when an ancestral receptor diverged to the modern-day immunoglobulin and TCR. Other molecules that undergo rearrangement have not been described in vertebrates, nor have intermediates been identified that have features of both these gene families. We report here the isolation of a new member of the immunoglobulin superfamily from the nurse shark, Ginglymostoma cirratum, which contains one variable and five constant domains and is found as a dimer in serum.

Amino Acid Sequence↗