Tethering of soluble immune effectors to mucin and chitin reflects a convergent and dynamic role in gut immunity.

The immune system employs soluble effectors to shape luminal spaces. Antibodies are soluble molecules that effect immunological responses, including neutralization, opsonization, antibody-dependent cytotoxicity and complement activation. These molecules are comprised of immunoglobulin (Ig) domains. The N-terminal Ig domains recognize antigen, and the C-terminal domains facilitate their elimination through phagocytosis (opsonization). A less-recognized function mediated by the C-terminal Ig domains of the IgG class of antibodies (Fc region) involves the formation of multiple low-affinity bonds with the mucus matrix. This association anchors the antibody molecule to the matrix to entrap potential pathogens. Even though invertebrates are not known to have antibodies, protochordates have a class of secreted molecules containing Ig domains that can bind bacteria and potentially serve a similar purpose. The VCBPs (V region-containing chitin-binding proteins) possess a C-terminal chitin-binding domain that helps tether them to chitin-rich mucus gels, mimicking the IgG-mediated Fc trapping of microbes in mucus. The broad functional similarity of these structurally divergent, Ig-containing, secreted effectors makes a case for a unique form of convergent evolution within chordates. This opinion essay highlights emerging evidence that divergent secreted immune effectors with Ig-like domains evolved to manage immune recognition at mucosal surfaces in strikingly similar ways. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Eleven distinct VH gene families and additional patterns of sequence variation suggest a high degree of immunoglobulin gene complexity in a lower vertebrate, Xenopus laevis.

Lower vertebrate species, including Xenopus laevis, exhibit restricted antibody diversity relative to higher vertebrates. We have analyzed more than 180 VH gene-containing recombinant clones from an unamplified spleen cDNA library by selective sequencing of JH and CH positive clones following iterative hybridization screening with family-specific VH probes, 11 unique families of VH genes, each associated with a unique genomic Southern blot hybridization pattern, are described and compared. Considerable variation in the number of hybridizing components detected by each probe is evident. The nucleotide sequence difference between VH families is as great as, if not more than, that reported in other systems, including representatives of the mammalian, avian, and elasmobranch lineages. Some Xenopus Ig gene families encode alternative amino acids at positions that are otherwise invariant or very rarely substituted in known Igs. Furthermore, variations in complementarity determining region sequences among members of the same gene family and high degrees of DH and JH region complexity are described, suggesting that in at least this lower vertebrate species, the diversity of expressed Ig VH genes is not restricted.

Somatic variation precedes extensive diversification of germline sequences and combinatorial joining in the evolution of immunoglobulin heavy chain diversity.

In Heterodontus, a phylogenetically primitive shark species, the variable (VH), diversity (DH), joining (JH) segments, and constant (CH) exons are organized in individual approximately 18-20-kb "clusters." A single large VH family with > 90% nucleic acid homology and a monotypic second gene family are identified by extensive screening of a genomic DNA library. Little variation in the nucleotide sequences of DH segments from different germline gene clusters is evident, suggesting that the early role for DH was in promoting junctional diversity rather than contributing unique coding specificities. A gene-specific oligodeoxynucleotide screening method was used to relate specific transcription products (cDNAs) to individual gene clusters and showed that gene rearrangements are intra- rather than intercluster. This provides further evidence for restricted diversity in the immunoglobulin heavy chain of Heterodontus, from which it is inferred that combinatorial diversity is a more recently acquired means for generating diversity. The observed differences between cDNA sequences selected and the sequences of segmental elements derived from conventional genomic libraries as well as from VH segment-specific libraries generated by direct PCR amplification of genomic DNA indicate that the VH repertoire is diversified by both junctional diversity and somatic mutation. Taken together, these findings suggest a heretofore unrecognized contribution of somatic variation that preceded both extensive diversification of the germline repertoire and the combinatorial joining process in the evolution of humoral immunity.

Generation of immunoglobulin light chain gene diversity in Raja erinacea is not associated with somatic rearrangement, an exception to a central paradigm of B cell immunity.

In all vertebrate species examined to date, rearrangement and somatic modification of gene segmental elements that encode portions of the antigen-combining sites of immunoglobulins are integral components of the generation of antibody diversity. In the phylogenetically primitive cartilaginous fishes, gene segments encoding immunoglobulin heavy and light chain loci are arranged in multiple clusters, in which segmental elements are separated by only 300-400 bp. In some cases, segmental elements are joined in the germline of nonlymphoid cells (joined genes). Both genomic library screening and direct amplification of genomic DNA have been used to characterize at least 89 different type I light chain gene clusters in the skate, Raja. Analyses of predicted nucleotide sequences and predicted peptide structures are consistent with the distribution of genes into different sequence groups. Predicted amino acid sequence differences are preferentially distributed in complementarity-determining versus framework regions, and replacement-type substitutions exceed neutral substitutions. When specific germline sequences are related to the sequences of individual cDNAs, it is apparent that the joined genes are expressed and are potentially somatically mutated. No evidence was found for the presence of any type I light chain gene in Raja that is not germline joined. The type I light chain gene clusters in Raja appear to represent a novel gene system in which combinatorial and junctional diversity are absent.

Spontaneous autoimmunization to GIX cell surface antigen in hybrid mice.

The GIX antigen expressed on the thymocytes of GIX+ mice is a type-specific constituent of glycoprotein gp70, which forms the major envelope component of murine leukemia virus. In the prototype GIX+ mouse strain 129, this glycoprotein is a Mendelian character expressed independently of virus production. In the intact thymocyte plasma membrane, part of this glycoprotein, bearing group-specific (gs) antigen, is inaccessible to antibody. The moiety bearing the type-specific GIX determinant is accessible to GIX antibody, which may be an important factor in determining the consequences of autoimmune responses involving GIX. Previously, all attempts to induce GIX antibody in mice had failed. We now find that the hybrid mouse (B6-GIX+ X 129) spontaneously produces substantial amounts of GIX antibody, presumably of the IgM class appearing as early as 2 mo of age. The specificity of the GIX natural mouse antibody is the same as that recognized by the conventional GIX typing serum produced in rats ("anti-NTD"). As neither parent strain produces appreciable GIX antibody, we surmise that this autoimmune response requires two dominant genes, each parent contributing a high-response allele to the hybrid. These can be envisaged as two immune response loci, controlling different immunocompetent cells which must cooperate to produce GIX antibody. Production of GIX antibody by the hybrids increases progressively with age. This is accompanied by decreased expression of GIX antigen on their thymocytes. We attribute this to antigenic modulation. Antibody to gs antigen of gp70 is also found in autoimmune (B6-GIX+ X 129) hybrids but not in either parent strain. We are investigating evidence of a pathological autoimmune syndrome in these hybrids. The special interest of this syndrome is that it presumably signifies the consequences of autoimmunization to a single C-type virus component, expressed without significant virus production, in a mouse with no evident genetic predisposition to such disease in the absence of that antigen.

Hv(1), a variable-region genetic marker of human immunoglobulin heavy chains.

A new antigenic determinant was discovered with a hemagglutination-inhibition assay system. Designated Hv(1), it is located in the variable region of human immunoglobulin heavy chains of the G, M, and A classes. Pedigree and population analyses suggest that it has an autosomal dominant mode of inheritance. This represents the first description of an allotypic determinant in the variable region of human immunoglobulins.

Analysis of lipophilic carcinogen-membrane interactions using a model human erythrocyte membrane system.

Human erythrocytes have been used as a model for evaluating the chemical carcinogen-plasma membrane interaction. The carcinogenic aromatic amines 2-acetylaminofluorene, dimethylaminoazobenzene, and 3'-methyldimethylaminoazobene stabilize erythrocytes against lysis in hypotonic solution. In general, the stabilization potential of these compounds reflects their oil:water partition coefficients and may be related to both their extracellular distribution and ultimate capacity for penetration of target cells. The polycyclic aromatic hydrocarbons, 3-methylcholanthrene and benz[a]anthracene, confer little protection against hemolysis and simultaneous incubation of nonprotective 3-methylcholanthrene and protective 3'-methyldimethylaminoazobenzene slightly alters the stabilization afforded by the latter. 7,12-dimethylbenz[a]anthracene exhibits greater protective capacity than does benz[a]anthracene. Polycyclic aromatic hydrocarbons manifested considerably higher degrees of absolute binding to erythrocytes in isotonic solution than did aromatic amines. The difference in erythrocyte binding and stabilization exhibited by the 2 classes of carcinogens suggest distinct mechanisms of membrane association that may relate to their metabolic disposition.

Human Txk: genomic organization, structure and contiguous physical linkage with the Tec gene.

Txk is a Tec-family tyrosine kinase expressed in mouse and human T lymphocytes. Among the Tec kinases, Txk is unique in that its amino terminal region does not include a pleckstrin homology domain or other known extended functional region. Txk is encoded at human chromosome 4p12 and at a recognized region of conserved synteny on mouse chromosome 5. The genomic organization of Txk consists of 15 exons with strong exon-intron organizational homology to Btk, the only other Tec-family kinase for which the genomic structure is fully known. The human Tec gene also maps to 4p12 and, based on limited studies reported here, possesses organizational homology with Btk and Txk. We have sequenced a continuous region of DNA that contains 3' Tec and 5' Txk exons separated by only a approximately 1.5 kb intergenic region containing the putative promoter region of Txk. The close physical linkage of these Tec-family tyrosine kinases, which are expressed in different hematopoetic cell lineages, suggests their potential for coordinate cis-regulation.

Differential perturbation of erythrocyte membrane function by structurally related polycyclic aromatic hydrocarbons.

Examination of the interaction of a number of structurally related polycyclic aromatic hydrocarbons with the erythrocyte plasma membrane indicated that the presence and position of methyl groups on the lipophilic hydrocarbon nucleus determined whether the compound acted as an inhibitor of membrane function. 7,12-Dimethylbenz(a)anthracene, a potent carcinogen, acted as a noncompetitive inhibitor of membrane acetylcholinesterase. The inhibition depended on the anion composition of the buffer at the time of exposure of the cells to inhibitor, i.e., it was only manifest in the presence of an anion gradient. The temperature dependence of the intact cell enzyme in the presence of inhibitor was influenced by the temperature at which the compound was added prior to assay and may involve the perturbation of tightly associated lipids. Glucose exchange across the membrane was inhibited by the same compounds which inhibit acetylcholinesterase. The temperature dependence of the exchange was not grossly altered by the presence of 7,12-dimethylbenz(a)anthracene. The observed inhibition of two membrane functions by the polycyclic aromatic hydrocarbons does not correlate simply with their theoretical octanol/water partition coefficients, water solubilities, or ability to confer membrane stabilization against osmotic hemolysis. This demonstration of differential inhibition by compounds having the same overall hydrophobicity was unexpected and suggests a more complex mode of interaction with the cell membrane.

Investigations of the molecular basis for the temperature-dependent insolubility of cryoglobulins. VI. Quenching by acrylamide of the intrinsic tryptophan fluorescence of cryoglobulin and non-cryoglobulin IgM proteins.

The acrylamide-quenching patterns of the intrinsic tryptophan fluorescence of six cold-soluble monoclonal immunoglobulin M (IgM) and two monoclonal IgM proteins possessing cryoglobulin properties (abnormal cold insolubility) have been compared. Static and dynamic components of quenching have been resolved by a modified form of the Stern-Volmer relationship. The unusual observation of static quenching seen with the multitryptophan containing IgM is determined to be a consequence of essentially homogeneous indole fluorescence arising from conserved tryptophan residues within each homologous immunoglobulin domain. Although the static component of the quenching of the two IgM cryoimmunoglobulins examined is similar to that of the non-cryoimmunoglobulin, IgM, some of the cryoglobulin's tryptophan residues appear to be more kinetically exposed to acrylamide than the tryptophans in the non-cryoglobulin IgM. An unusually large negative entropy of activation observed for the quenching process of both cryoimmunoglobulins suggests some abnormality in the dynamic (flexibility) properties of these proteins.

Differential perturbation of erythrocyte membrane-associated transport and enzyme activities by structurally related lipophilic compounds.

The alteration of two erythrocyte plasma membrane functions, acetylcholine hydrolysis and glucose exchange, by a series of structurally related small lipophilic compounds which exhibit antihemolytic behavior was studied. 2-Methyldimethylaminoazobenzene is a more potent inhibitor of acetylcholinesterase than the 3'-methyl analogue, while the unsubstituted compound fails to inhibit. Esterase inhibition by the 2-methyl compound is non-competitive and dependent on the anion composition of the assay buffer. The temperature dependence of acetylcholinesterase activity in the presence of the 2-methyl compound suggests that interaction with inhibitor is influenced by the state of lipids tightly bound to the enzyme. Glucose exchange is inhibited to the same extent by both methyl derivatives but not by the unsubstituted dye, and the temperature dependence in the presence of inhibitor is not grossly altered. The lack of correlation between inhibition of membrane function adn stabilization of erythrocytes against osmotic hemolysis is discussed.

Raman spectra and conformational structures of Fab mu and (Fc)5 mu fragments of cryoglobulin IgM-kappa McE.

Raman spectra have been obtained on aqueous solutions of the following human immunoglobulins: IgM-kappa McE, IgG-kappa Ger, IgM-kappa WSm and IgG-lambda Gui. The former two species exhibit the property of cryoprecipitation. Comparison of the spectra shows that all immunoglobulins have similar secondary structures, predominantly of the beta-sheet type. Fab mu and (Fc)5 mu fragments of IgM-kappa McE also yield Raman spectra which indicate closely similar secondary structures. Minor differences among the spectra can be explained by differences in amino acid compositions of the respective proteins.

Analysis of polypeptide disposition in human erythrocyte membranes employing membrane inversion.

High resolution segregation of erythrocyte membrane polypeptides achieved by isoelectric focusing in 8 M urea was employed in conjunction with surface-restricted radioiodination to analyze the disposition of polypeptides within the human erythrocyte membrane. Several membrane polypeptides showed significant uptake of radioiodine, with the principal labeled component migrating between pH values of 3.0 and 3.5. Two approaches were taken in examining membrane polypeptide disposition on both faces of the erythrocyte membrane. Saturation labeling of the outer face of the membrane with one iodine isotope followed by cell lysis and reiodination with a second iodine isotope did not prove feasible and another procedure based on surface iodination with 125-I, formation of sealed inside-out vesicles and re-iodination with 131-I was adopted. Studies of sialic acid release from the membrane surface and trypsin cleavage of radioiodinated peptides indicated that selectively labeled, sealed inside-out vesicles had been formed. The ratio of 125-I to 131-I in membrane polypeptides separated by isoelectric focusing confirmed the existence of externally disposed, internally disposed and spanning proteins.

Immune-type diversity in the absence of somatic rearrangement.

Immunoglobulin gene diversity has been characterized to varying degrees in modern representatives of all of the major radiations of cartilaginous fish. A pattern of overall chromosomal relationships of the various types of joined and unjoined Ig gene clusters is suggested in which the essential features are: (a) both Ig heavy and light-chain gene clusters occur on multiple chromosomes, (b) various classes of Ig are interspersed, (c) not all individual gene loci appear to be closely linked (Fig. 2). The cluster-type Ig gene system appears to be a series of (potentially) individually regulated loci analogous in part to the olfactory receptor gene system (BUCK and AXEL 1991) and markedly distinct from Ig loci in other vertebrate groups and TCR genes. Such a system would be ideal for the creation of variation in both form and function in a large number of clusters while preserving or partially preserving specificity in a number of other gene clusters. The full range of joined genes and the relative number of joined genes (as relates to unjoined genes), have yet to be determined. Nevertheless, a number of conclusions can be drawn: (a) four distinct forms of heavy-chain joining have been identified (VDD-J, VD-DJ, V-D-DJ, and VDJ; Fig. 1); (b) light-chain genes, which possess only two recombining elements, can be found in either unjoined (V-J) or joined (VJ) forms (Fig. 1); (c) physical linkage between individual joined and unjoined genes has not been established, although such investigations have not been pursued in a significantly rigorous manner as to rule out this possibility; (d) joined light-chain genes are expressed and can be somatically mutated. Can germline joining be viewed as an ancestral character? The answer to this needs to be considered in the context of an overall system in which the level of structural and functional redundancy is extremely high. Joining is an adaptation that is unique to multicluster gene families. The phenomenon overcomes the possibility of not generating a specific form of a receptor, a major shortcoming of conventional rearranging Ig and TCR gene systems. The limitation of encoding specific receptors is compensated through large numbers of additional gene clusters that retain the capacity to rearrange and generate new specificities. Commitment of a V region to diverse, fixed specificity also is a property of the NITR genes, which although not related closely to Ig in a structural sense, may reflect an analogous phenomena. The possibility that immune-type diversity is achieved in the absence of somatic rearrangement and that remnants of such systems could be operative in immune recognition in contemporary vertebrates is of extraordinary significance in terms of our overall understanding of the relationships between adaptive and innate immune recognition.

Molecular basis for the temperature-dependent insolubility of cryoglobulins--XI. Sequence comparison of the heavy-chain variable regions of the human cryoimmunoglobulins McE and Hil by metric analysis.

The amino acid sequences of the VH-domains from two human cryoimmunoglobulins have been compared with one another and with the VH-domains of noncryoglobulins by the mathematical method of metric analysis. The VHII sequence of McE [Gerber-Jenson et al. (1981), J. Immun. 126, 1212-1216] and the VHIII sequence of Hil [Chiu et al (1979), Biochemistry, 18, 553-560] resemble much more closely the VH-sequences of noncryoglobulins from their own subgroups than they resemble one another. Neither cryoglobulin sequence contains an unusual insertion or deletion of residues. Based on the crystallographic structure of the VHII domain in the Fab fragment of the human noncryoglobulin Newm [Saul et al. (1978), J biol. Chem., 253, 585-597], McE and Hil each contain two unprecedented residues in the outer beta-sheet structure of the VH-domain. The inwardly directed sidechains of Gly-71 and Ile-84 in McE may perturb the internal hydrophobic interactions and normal folding of adjacent segments of the outer beta-sheet from the third framework region. In contrast, the outwardly directed sidechains of Ile-23 and Arg-77 in Hil may perturb the external hydrophilic properties and folding of adjacent segments of the outer beta-sheet from the first and third framework regions. Thus, the monoclonal immunoglobulins McE and Hil may display cold-induced insolubility by different perturbations of the outer surface of the VH-domain. In each case, the unprecedented framework residues that mnay be responsible could have arisen by two point mutations involving single base changes.

The zebrafish fth1, slc3a2, men1, pc, fgf3 and cycd1 genes define two regions of conserved synteny between linkage group 7 and human chromosome 11q13.

In addition to being an excellent model system for studying vertebrate development, the zebrafish has become a great tool for gene discovery by mutational analysis. The recent availability of the zebrafish EST database and radiation hybrid mapping panels has dramatically expanded the framework for genomic research in this species. Developing comparative maps of the zebrafish and human genomes is of particular importance for zebrafish mutagenesis studies in which human orthologs are sought for zebrafish genes. However, only partial cDNA sequences are determined routinely for mapped ESTs, leaving the identity of the EST in question. It previously had been reported that zebrafish linkage group 7 shares conserved synteny with human chromosome 11q13. In an effort to further define this relationship, five full-length zebrafish cDNAs, fth1, slc3a2, prkri, cd81, and pc, as well as one putative human gene, DBX were identified and their map positions ascertained. These six genes, along with men1, fgf3 and cycd1 define two regions of conserved synteny between linkage group 7 and 11q13.

Generation of a P1 artificial chromosome library of the Southern pufferfish.

We describe the generation of a P1 artificial chromosome genomic library from the Southern pufferfish, Spheroides nephelus. The arrayed library consists of approximately 30,000 clones and has an average insert size of 125-150 kb. The coverage is estimated to encompass seven to eight genome equivalents. The library has been used for isolating numerous genomic clones and for establishing contigs of several multigene families. Analysis of several of the clones from this library suggests a preponderance of CA repeat tracts relative to their abundance in humans. The library and high-density filters have been made available to the scientific public through genomics distribution companies.

Putative immunoglobulin VH genes of the goldfish, Carassius auratus, detected by heterologous cross-hybridization with a murine VH probe.

By using a defined cDNA probe for the VH region of a murine phosphocholine-binding myeloma protein (S107) we have defined a family of distinct cross-hybridizing DNA sequences in genomic DNA of the goldfish. The estimated number of the goldfish putative VH family detectable by the S107 probe is about 36. By using two putative goldfish VH probes to analyze, by hybridization, the relationships among seven of the goldfish genomic clones, we have determined that the putative goldfish VH genes recognized by the S107 probe comprise at least several distinct families that are not closely related.

Monoclonal cryoglobulinemia with macroglobulinemia in a dog.

A 9-year old female Doberman Pinscher had a 5-month history of epistaxis. On routine testing of the dog's serum, blockage of the intake tubes of the equipment resulted. At this point, cryoglobulinemia was suspected and was later confirmed to be due to a monoclonal cryoglobulin (IgM), by means of immunochemical studies. Clinical recovery was achieved by use of cytotoxic drugs.