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.     

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.     

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.     

DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains

The covalent rejoining of DNA ends at single-stranded or double-stranded DNA breaks is catalyzed by DNA ligases. Four DNA ligase activities (I-IV) have been identified in mammalian cells [1]. It has recently been demonstrated that DNA ligase IV interacts with and is catalytically stimulated by the XRCC4 protein [2,3], which is essential for DNA double-strand break repair and the genomic rearrangement process of V(D)J recombination [4]. Together with the finding that the yeast DNA ligase IV homologue is essential for nonhomologous DNA end joining [5-7], this has led to the hypothesis that mammalian DNA ligase IV catalyzes ligation steps in both of these processes [8]. DNA ligase IV is characterized by a unique carboxy-terminal tail comprising two BRCT (BRCA1 carboxyl terminus) domains. BRCT domains were initially identified in the breast cancer susceptibility protein BRCA1 [9], but are also found in other DNA repair proteins [10]. It has been suggested that DNA ligase IV associates with XRCC4 via its tandem BRCT domains and that this may be a general model for protein-protein interactions between DNA repair proteins [3]. We have performed a detailed deletional analysis of DNA ligase IV to define its XRCC4-binding domain and to characterize regions essential for its catalytic activity. We find that a region in the carboxy-terminal tail of DNA ligase IV located between rather than within BRCT domains is necessary and sufficient to confer binding to XRCC4. The catalytic activity of DNA ligase IV is affected by mutations within the first two-thirds of the protein including a 67 amino-acid amino-terminal region that was previously thought not to be present in human DNA ligase IV [11].     

The great escape: structure and function of the autotransporter proteins

The autotransporters, a family of secreted proteins from Gram-negative bacteria, possess an overall unifying structure comprising three functional domains: the amino-terminal leader sequence, the secreted mature protein (passenger domain) and a carboxy-terminal (beta-) domain that forms a beta-barrel pore to allow secretion of the passenger protein. Members of this family have been implicated as important or putative virulence factors in many Gram-negative pathogens.     

Functional interactions between isolated SH2 domains and insulin/Ras signaling pathways of Xenopus oocytes: opposite effects of the carboxy- and amino-terminal SH2 domains of p85 PI 3-kinase

Purified amino-terminal Src homology 2 (SH2) domains of GAP, PLCgamma1 and the p85alpha subunit of PI 3-kinase, as well as the carboxy-terminal SH2 domain of the latter protein and the unique SH2 domain of Grb2, were injected into full grown, stage VI Xenopus laevis oocytes. None of the injected domains showed any effect when injected alone, nor did they affect the rate of GVBD induced by progesterone, an adenylate cyclase-dependent process. On the other hand, the unique Grb2 SH2 domain and all N-terminal SH2 domains injected inhibited to various degrees the rate of insulin-induced GVBD, a tyrosine kinase dependent pathway. Interestingly, and in contrast to the behavior shown by the N-terminal domain of the same molecule, the C-terminal SH2 domain of p85 did not inhibit, but slightly accelerated the rate of GVBD induced by insulin. Furthermore, whereas the Grb SH2 domain and all N-terminal SH2 domains tested failed to co-operate with normal Ras protein to induce GVBD, the C-terminal SH2 domain of p85alpha exhibited significant synergy when coinjected with normal Ras protein, indicating that the C- and N-terminal SH2 domains of p85alpha exert opposite (positive and negative, respectively) regulatory roles in the control of oocyte insulin/Ras signaling pathways. Our results demonstrate that the purified, isolated SH2 domains retain structural and functional specificity and that Xenopus oocytes constitute an useful biological system to analyse their functional role in tyrosine kinase signaling pathways.     

Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells

Immunoglobulin (Ig)M+IgD+ B cells are generally assumed to represent antigen-inexperienced, naive B cells expressing variable (V) region genes without somatic mutations. We report here that human IgM+IgD+ peripheral blood (PB) B cells expressing the CD27 cell surface antigen carry mutated V genes, in contrast to CD27-negative IgM+IgD+ B cells. IgM+IgD+CD27(+) B cells resemble class-switched and IgM-only memory cells in terms of cell phenotype, and comprise approximately 15% of PB B lymphocytes in healthy adults. Moreover, a very small population (<1% of PB B cells) of highly mutated IgD-only B cells was detected, which likely represent the PB counterpart of IgD-only tonsillar germinal center and plasma cells. Overall, the B cell pool in the PB of adults consists of approximately 40% mutated memory B cells and 60% unmutated, naive IgD+CD27(-) B cells (including CD5(+) B cells). In the somatically mutated B cells, VH region genes carry a two- to threefold higher load of somatic mutation than rearranged Vkappa genes. This might be due to an intrinsically lower mutation rate in kappa light chain genes compared with heavy chain genes and/or result from kappa light chain gene rearrangements in GC B cells. A common feature of the somatically mutated B cell subsets is the expression of the CD27 cell surface antigen which therefore may represent a general marker for memory B cells in humans.     

Functional analysis of a rickettsial OmpA homology domain of Shigella flexneri icsA

Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into "actin tails" at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the beta domain), such that the amino-terminal 706 amino acid residues (the alpha domain) are exposed on the exterior of the bacillus. The alpha domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the alpha domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.     

Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains

The ERM proteins--ezrin, radixin, and moesin--occur in particular cortical cytoskeletal structures. Several lines of evidence suggest that they interact with both cytoskeletal elements and plasma membrane components. Here we described the properties of full-length and truncated radixin polypeptides expressed in transfected cells. In stable transfectants, exogenous full-length radixin behaves much like endogenous ERM proteins, localizing to the same cortical structures. However, the presence of full-length radixin or its carboxy-terminal domain in cortical structures correlates with greatly diminished staining of endogenous moesin in those structures, suggesting that radixin and moesin compete for a limiting factor required for normal associations in the cell. The results also reveal distinct roles for the amino- and carboxy-terminal domains. At low levels relative to endogenous radixin, the carboxy-terminal polypeptide is associated with most of the correct cortical targets except cleavage furrows. In contrast, the amino-terminal polypeptide is diffusely localized throughout the cell. Low level expression of full-length radixin or either of the truncated polypeptides has no detectable effect on cell physiology. However, high level expression of the carboxy-terminal domain dramatically disrupts normal cytoskeletal structures and functions. At these high levels, the amino-terminal polypeptide does localize to cortical structures, but does not affect the cells. We conclude that the behavior of radixin in cells depends upon activities contributed by separate domains of the protein, but also requires modulating interactions between those domains.     

The variable domain of nonassembled Ig light chains determines both their half-life and binding to the chaperone BiP

Not much is known about the features that determine the biological stability of a molecule retained in the endoplasmic reticulum (ER). Ig light (L) chains that are not secreted in the absence of Ig heavy (H) chain expression bind to the ER chaperone BiP as partially folded molecules until they are degraded. Although all Ig L chains have the same three-dimensional structure when part of an antibody molecule, the degradation rate of unassembled Ig L chains is not identical. For instance, the two nonsecreted murine Ig L chains, kappaNS1 and lambdaFS62, are degraded with half-lives of approximately 1 and 4 hr, respectively, in the same NS1 myeloma cells. Furthermore, the BiP/lambdaFS62 Ig L chain complex appears to be more stable than the BiP/kappaNS1 complex. Here, we used the ability of single Ig domains to form an internal disulfide bond after folding as a measure of the folding state of kappaNS1 and lambdaFS62 Ig L chains. Both of these nonsecreted L chains lack the internal disulfide bond in the variable (V) domain, whereas the constant (C) domain was folded in that respect. In both cases the unfolded V domain provided the BiP binding site. The stability of BiP binding to these two nonsecreted proteins was quite different, and both the stability of the BiP:Ig L chain complex and the half-life of the Ig L chain could be transferred from one Ig L chain isotype to the other by swapping the V domains. Our data suggest that the physical stability of BiP association with an unfolded region of a given light chain determines the half-life of that light chain, indicating a direct link between chaperone interaction and delivery of partially folded substrates to the mammalian degradation machinery.     

An endogenous sialoprotein and a bacterial B cell superantigen compete in their VH family-specific binding interactions with human Igs

Staphylococcal protein A (SpA), a bacterial membrane protein, and protein Fv (Fv binding protein (pFv)), a human sialoprotein involved in gut-associated immunity, have both recently been shown to have unconventional V(H) family-restricted binding interactions with Igs. To determine whether these Ig binding proteins interact with related structures, we performed a series of comparative binding studies. The results confirmed that both molecules are bound by most V(H)3 IgM, but pFv is also recognized by V(H)3 and V(H)6 Ig that do not interact with SpA. We discovered that pFv and SpA (or a single domain of SpA) can compete for binding to a V(H)3 Ab, which suggests that they can recognize the same (or adjacent) V(H) sites. For both SpA and pFv, binding is less frequent among IgG than IgM. However, V(H)3 IgG more commonly possess Fab-mediated binding activity for pFv than for SpA. Binding studies of denatured Ig suggested that both pFv and SpA interact with conformationally dependent V(H) sites, although in certain cases pFv binding is more permissive than SpA binding. Taken together, these results indicate that the superantigen properties of SpA, a microbial protein, and those of pFv, an endogenous sialoprotein, involve binding interactions with overlapping and at times functionally equivalent sites in the V(H) domain, indicating that self and foreign proteins can employ highly conserved strategies to create superantigens for the Ag receptors of B lymphocytes.     

Structure and function of HIV-1 and SIV Tat proteins based on carboxy-terminal truncations, chimeric Tat constructs, and NMR modeling

To further define the structure and function of the domains in HIV-1 and SIV Tat proteins, chimeric Tat cDNA expression constructs were generated with crossover points at the carboxy-terminal end of the cysteine rich domain. The chimera containing the amino-terminal region of SIV and carboxy-terminal region of HIV exhibited activity similar to HIV-1 Tat and SIV Tat on both the HIV-1 and SIV LTRs. In contrast, the reciprocal chimera functioned poorly. As determined by the activity of carboxy-terminal truncation mutants, the region immediately downstream of the basic domain is critical for efficient transactivation by HIV-1 Tat, but not SIV Tat protein. In this report, we present a model for Tat domains based on NMR data and the known functional properties of Tat protein. According to our modeling two sites for protein : protein interactions are present in HIV-1 and SIV Tat proteins. Site I, which is presumably involved in cyclin T binding, is similar in both HIV-1 and SIV Tat proteins as well as in Tat chimeras. Site II, however appears structurally different in HIV-1 and SIV Tat models, although in both cases is comprised of amino and carboxy-terminal residues. Differences in Site II may thus account for the differential activities of HIV-1 and SIV Tat carboxy-terminal truncations. Site II in the poorly active chimera differs significantly from that found in HIV-1 and SIV Tat proteins. The two site structural model presented here may have important implications for the role of Tat in HIV pathogenesis and may provide insights for the design of Tat vaccines and targeted therapeutics.     

Pairing of variable heavy and variable kappa chains in individual naive and memory B cells

A functional Ig consists of two heterodimers each of which is composed of a heavy and a light chain. Although there is increasing knowledge about the events that govern the rearrangement of the genes encoding each individual chain, only very limited information is available about the mechanisms governing the pairing of variable heavy (V(H)) and variable light (V(L)) chains. Using a single cell PCR, we were able to obtain V(H) and Vkappa chains from 144 individual human CD19+/IgM+ B cells. Pairing of specific V(H) or Vkappa families was not observed, nor was the length or the amino acid composition of the CDR3s of V(H) and Vkappa chains in individual B cells similar. Comparison of V(H) and Vkappa genes in B cells in which one or both contained evidence of somatic hypermutation with those with no mutations revealed a significant decrease in the mean length of the V(H) CDR3. Moreover, there was a significant correlation between the frequencies of mutations in V(H) and Vkappa gene pairs in individual B cells. These results indicate that Ag-mediated selection as opposed to V(H)DJ(H) recombination or subsequent Ig chain pairing tended to approximate the CDR3 lengths and the frequency of mutations of V(H) and Vkappa in individual B cells.     

Domain structure of the ATP-binding-cassette protein MalK of salmonella typhimurium as assessed by coexpressed half molecules and LacK'-'MalK chimeras

ATP-binding-cassette (ABC) subunit MalK of the binding protein-dependent transport system for maltose of Salmonella typhimurium and Escherichia coli is crucial to the transport process but also exhibits a repressing activity on other genes of the maltose regulon. The latter function has been attributed to a carboxy-terminal extension by which MalK differs in length from a prototype ABC protein. In order to define the boundaries of putative functional domains of MalK, we have analyzed pairs of N- and C-terminally truncated MalK proteins of S. typhimurium. Coexpressed half molecules of about equal lengths (MalKN1: residues 1 to 179; MalKC1: residues 179 to 369) restored the transport activity of a malK strain and displayed substantial regulatory activity. The same regulatory activity was obtained when malKC1 was expressed separately. These results indicate that a covalent linkage is not absolutely essential for function and that the protein might be composed of two structurally distinct entities. To elucidate further the minimal structural requirements for the regulatory function of MalK, we have studied chimeric proteins that have C-terminal portions of MalK fused to the corresponding amino-terminal fragments of its close homolog LacK. Functional analyses revealed that a fusion containing only the C-terminal extension of MalK (Q263 to V369) is sufficient to display half-maximal regulatory activity. This activity increased with the lengths of the MalK portions present in the chimeras. Furthermore, the failure of two chimeras to support maltose transport suggests a structurally critical region between residues 243 and 264. In the absence of a crystal structure, this work contributes to the understanding of the multiple functions of MalK.     

A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors

This paper describes the identification of a new family of mammalian genes that encode secreted proteins containing homology to the cysteine-rich ligand-binding domain found in the frizzled family of transmembrane receptors. The secreted frizzled-related proteins (sFRPs) are approximately 30 kDa in size, and each contains a putative signal sequence, a frizzled-like cysteine-rich domain, and a conserved hydrophilic carboxy-terminal domain. The sFRPs are not the products of differential splicing of the known frizzled genes. Glycosylphosphatidylinositol-anchored derivatives of sFRP-2 and sFRP-3 produced in transfected human embryonic kidney cells confer cell-surface binding by the Drosophila Wingless protein. These observations suggest that sFRPs may function in vivo to modulate Wnt signaling, or, alternatively, as novel ligands for as yet unidentified receptors.