Scavenger Receptor Cysteine-rich Research Articles

The mouth: a gateway or a trap for HIV?

Mucosal sites represent the primary routes of HIV transmission, yet the oral mucosa appears uniquely resistant to HIV-1 infection. Since the oral cavity is not conducive to either infection or transmission of HIV, characterizing the responsible resistance factors, particularly components of innate immunity, and their mechanisms of action may identify new opportunities for interference with viral acquisition at other mucosal sites. Although many of the innate immune factors described to date exhibit both antibacterial and antiviral activities, molecules that inhibit HIV-1 are largely HIV-1 specific. Nonetheless, if these oral innate factors can be identified, characterized, and harnessed, they may be useful in protection and/or intervention in acute HIV-1 infection. Endogenous molecules acting independently or synergistically, given their lack of substantive side effects, may represent an alternative novel approach for reducing HIV risk and suppressing infection. While HIV infection via breast milk does involve oral trafficking and is an important area of study, it will only be briefly discussed here. The major emphasis of this article is on the oral cavity and salivary substances that can influence HIV infection Epidemiology of oral transmission of HIV More than twenty-five years into the global HIV/AIDS epidemic, it is well established that in addition to blood, the reproductive and/or rectal mucosae represent the primary portals of HIV entry in adults. There is less definitive evidence regarding whether other mucosal sites serve as viral conduits and in particular, uncertainty remains as to the possibility of transmission of HIV-1 via the oral entrance to the gastrointestinal (GI) tract. This is not a trivial concern, since despite the successes of combination therapy for HIV/AIDS, infection rates remain high, compounded by persistence of latent viral reservoirs, antiretroviral drug resistance, and the lack of an effective vaccine or potential cure. Although it is often accepted that oral transmission is of little or no consequence, we have failed to capitalize on that information to identify the mechanism(s) of protection at the oral mucosal surface. These mechanisms could provide clues for novel strategies of prevention/intervention at other more vulnerable viral entry sites. Early in the epidemic, evidence of the presence of HIV-1 in the oral cavity suggested the possibility of transmissibility via this route [1, 2]. Whereas in some early studies [3–7], oral transmission could not be established, a few studies implicated this route as a potential, even likely mode of transmission (e.g., [8]). In larger cohorts of individuals and in well-designed studies to monitor oral transmission, the rarity of HIV-1 infection by this route was substantiated [9]. Nonetheless, potential shortcomings in these studies, including accurately documenting the definitive route of infection, underscore our lack of certainty regarding the relative risk of oral transmission. (Reviews: [10, 11]). The low frequency of documented oral transmission of HIV-1 could reflect low titers of infectious virus in saliva. While high levels of HIV-1 RNA and antigens can routinely be measured in oral fluids, infectious virus was not detected in early studies attempting to isolate infectious virus from saliva [12, 13] or more recently in large cohort studies, despite plasma viremia [14, 15].

Activation of Rat Liver Microsomal Glutathione Transferase by Hepsin

Rat liver microsomal glutathione transferase (MGST1) is activated by limited proteolysis. Recently we purified a protease, hepsin, from rat liver microsomes that activates MGST1. In the present study the mechanism of MGST1 activation by hepsin was investigated. When MGST1 and hepsin were incubated at room temperature, MGST1 activity was markedly increased and the increase was decreased to the control level by further incubation with disulfide bond reducing agent dithiothreitol. MGST1 dimer was detected by electrophoresis after treatment of MGST1 with hepsin, instead of proteolytic product. MGST1 dimer formation accompanied by an increase in MGST1 activity was observed even in the presence of the protease inhibitor benzamidine. Furthermore, prolonged incubation of both enzymes caused the formation of MGST1 dimer and its proteolytic product. These results clearly show that the protease hepsin stimulates disulfide-linked MGST1 dimer formation resulting in activation of MGST1 and preferential degradation of MGST1 dimer. Since hepsin contains disulfide bonds in the scavenger receptor cysteine-rich (SRCR) domain, it was suggested that the SRCR domain interacts with MGST1 leading to thiol/disulfide exchange between the two enzymes followed by disulfide-linked MGST1 dimer formation.

Identification of the CD163 Protein Domains Involved in Infection of the Porcine Reproductive and Respiratory Syndrome Virus

Scavenger receptor CD163 is a key entry mediator for porcine reproductive and respiratory syndrome virus (PRRSV). To identify the CD163 protein domains involved in PRRSV infection, deletion mutants and chimeric mutants were created. Infection experiments revealed that scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR 5) is essential for PRRSV infection, while the four N-terminal SRCR domains and the cytoplasmic tail are not required. The remaining CD163 protein domains need to be present but can be replaced by corresponding SRCR domains from CD163-L1, resulting in reduced (SRCR 6 and interdomain regions) or unchanged (SRCR 7 to SRCR 9) infection efficiency. In addition, CD163-specific antibodies recognizing SRCR 5 are able to reduce PRRSV infection.

Antigenic basis of diversity in the γδ T cell co-receptor WC1 family

WC1 co-receptors are transmembrane glycoproteins with 11 extracellular scavenger receptor cysteine rich (SRCR) domains. They are related to the CD163 family but are uniquely expressed by γδ T cells. We recently showed that at least 13 members comprise the WC1 gene family in cattle, a model animal species for studies of γδ T cell biology. Since WC1 co-receptors participate in directing functional responses by γδ T cells either through the ligands they bind or the signals they transduce, availability of reagents to identify the expression of individual WC1 molecules of this diverse family would be valuable in further elucidating mechanisms of γδ T cell responsiveness. Although monoclonal antibodies (mAbs) have been widely used to identify WC1 co-receptors on γδ T cells, the locations of the antigenic epitopes recognized are unknown. Here, we mapped the epitopes to particular SRCR domains and evaluated their distribution among WC1 molecules. To do this, cDNA representing the extracellular domains of seven different WC1 genes was expressed in mammalian cells and analyzed for reactivity with anti-WC1 mAbs using ELISA and Western blotting. The study included mAbs that are broadly reactive with WC1 + γδ T cells and those that divide WC1 + γδ T cells into functionally distinct subpopulations. We found that mAb CC15 is a pan-reactive anti-WC1 mAb recognizing an epitope in the closely related SRCR domains 2 and 7 and that this epitope is present in at least domain 2 or 7 of all seven WC1 molecules evaluated here. Five other anti-WC1 mAbs, typified by mAb IL-A29, were found to be broadly reactive, recognizing epitopes in the related SRCR domains 4 and 9 but each having a unique pattern of reactivity with the seven WC1 molecules. Finally, the subpopulation-specific anti-WC1 mAbs, including those that recognize either the archetypal WC1.1 or WC1.2 molecule, were found to react with epitopes in the most variable WC1 domain, i.e. domain 1, of a restricted number of WC1 co-receptors.

Molecular cloning and analysis of SSc5D, a new member of the scavenger receptor cysteine-rich superfamily

Glycoproteins of the scavenger receptor cysteine-rich (SRCR) superfamily contain one or more protein modules homologous to the membrane-distal domain of macrophage scavenger receptor I. These domains can be found in the extracellular regions of membrane proteins and in secreted glycoproteins, from the most primitive species to vertebrates. A systematic, bioinformatics-based search for putative human proteins related to the forty-seven known human group B SRCR domains identified a new family member that we have called Soluble Scavenger with 5 Domains (SSc5D). SSc5D is a new soluble protein whose expression is restricted to monocytes/macrophages and T-lymphocytes, and is particularly enriched in the placenta. The gene encoding SSc5D spans 30 kb of genomic DNA, and contains fourteen exons producing a 4.8 kb-long mRNA. The mature polypeptide is predicted to consist of 1573 amino acids comprising, towards the N-terminus, five very similar SRCR domains that are highly conserved among non-marsupial mammals, and a large (>250 nm), very heavily glycosylated, mucin-like sequence towards the C-terminus. Each of the SRCR domains is encoded by a single exon, and contains eight cysteine residues, as observed for all other group B SRCR domains. A shorter isoform encoded by a weakly expressed, alternatively spliced transcript, which lacks the mucin-like C-terminal region, was also identified. It seems likely that SSc5D has a role at the interface between adaptive and innate immunity, or in placental function.

Secreted Cyclophilin A, a Peptidylprolyl cis-trans Isomerase, Mediates Matrix Assembly of Hensin, a Protein Implicated in Epithelial Differentiation

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

CD5 sweetens lymphocyte responses

The cell walls of pathogenic and commensal bacteria and fungi contain a variety of carbohydrate-containing moieties, not produced by animals. The presence of these lipopolysaccharides (LPSs), lipoteichoic acids (LTAs), peptidoglycans (PGNs), and zymosans in host tissues thus alerts the host to invasive bacterial or fungal infections. Several families of pattern recognition receptors (PRRs) expressed by epithelial and phagocytic cells contribute to recognition of microbial cell wall components. These PRRs include toll-like receptors (TLRs), nod-like receptors (NLRs), and scavenger receptors. Cell surface TLRs and cytosolic NLRs respond to microbial components by eliciting production of cytokines and chemokines that activate or amplify host inflammatory and immune responses. In contrast, at least some members of the scavenger-receptor cysteine-rich (SRCR) superfamily contribute more to the resolution of inflammation. In this issue of PNAS, a lymphocyte-expressed SRCR, CD5, is shown to bind the fungal polysaccharide β-glucan (1). Together with a recent study on CD6 (2), this work shows that SRCRs expressed on the lymphocyte cell surface are PRRs that detect diverse microbe-derived carbohydrates. Moreover, previous data implicating CD5 in negative regulation of lymphocyte antigen receptor signaling suggest that ligation of these PRRs with microbial components may profoundly influence lymphocyte activation and autoimmunity (Fig. 1).

Differential Binding of Inorganic Particles to MARCO

Alveolar macrophages (AM) in the lung have been documented to play pivotal roles in inflammation and fibrosis (silicosis) following inhalation of crystalline silica (CSiO(2)). In contrast, exposure to either titanium dioxide (TiO(2)) or amorphous silica (ASiO(2)) is considered relatively benign. The scavenger receptor macrophage receptor with collagenous structure (MARCO), expressed on AM, binds and internalizes environmental particles such as silica and TiO(2). Only CSiO(2) is toxic to AM, while ASiO(2) and TiO(2) are not. We hypothesize that differences in induction of pathology between toxic CSiO(2) and nontoxic particles ASiO(2) and TiO(2) may be related to their differential binding to MARCO. In vitro studies with Chinese hamster ovary (CHO) cells transfected with human MARCO and mutants were conducted to better characterize MARCO-particulate (ASiO(2), CSiO(2), and TiO(2)) interactions. Results with MARCO-transfected CHO cells and MARCO-specific antibody demonstrated that the scavenger receptor cysteine-rich (SRCR) domain of MARCO was required for particle binding for all the tested particles. Only TiO(2) required divalent cations (viz., Ca(+2) and/or Mg(+2)) for binding to MARCO, and results from competitive binding studies supported the notion that TiO(2) and both the silica particles bound to different motifs in SRCR domain of MARCO. The results also suggest that particle shape and/or crystal structure may be the determinants linking particle binding to MARCO and cytotoxicity. Taken together, these results demonstrate that the SRCR domain of MARCO is required for particle binding and that involvement of different regions of SRCR domain may distinguish downstream events following particle binding.

A common binding motif for various bacteria of the bacteria-binding peptide SRCRP2 of DMBT1/gp-340/salivary agglutinin

Abstract Salivary agglutinin (DMBT1SAG) is identical to lung glycoprotein-340 and encoded by deleted in malignant brain tumors-1. It is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, proteins that have one or more SRCR domains. Salivary agglutinin plays a role in oral innate immunity by the binding and agglutination of oral streptococci. S. mutans has been shown to bind to a 16-mer peptide (QGRVEVLYRGSWGTVC) located within the SRCR domains. Within this peptide, designated SRCR Peptide 2, residues VEVL and W were critical for binding. The aim of this study was to investigate binding of DMBT1SAG to other bacteria. Therefore, interaction between a series of bacteria and DMBT1(SAG), SRCR peptide 2 and its alanine substitution variants was studied in adhesion and agglutination assays. For different bacteria there was a highly significant correlation between adhesion to DMBT1SAG and adhesion to SRCR peptide 2 suggesting that SRCR peptide 2 is the major bacteria binding site. An alanine substitution scan showed that 8 amino acids were involved in binding (xRVEVLYxxSWxxxx). The binding motifs varied for different species were found, but the residues VxVxY and W were always present. In conclusion, a common binding motif (RVEVLYxxxSW) within the SRCR domains is responsible for the broad bacteria-binding spectrum of DMBT1SAG.

Towards the Definition of a Chimpanzee and Human Conserved CD6 Domain 1 Epitope Recognized by T1 Monoclonal Antibody

Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.

Genome Analysis of the Unicellular Green Alga Chlamydomonas reinhardtii Indicates an Ancient Evolutionary Origin for Key Pattern Recognition and Cell-Signaling Protein Families

The evolution of specific cell signaling and adhesion domains may have played an important role in the transition to a multicellular existence in the metazoans. Genomic analysis indicates that several signaling domains predominately found in animals are also present in the unicellular green alga, Chlamydomonas reinhardtii. A large group of proteins is present, containing scavenger receptor cysteine-rich (SRCR) and C-type lectin domains, which function in ligand binding and play key roles in the innate immune system of animals. Chlamydomonas also contains a large family of putative tyrosine kinases, suggesting an important role for phosphotyrosine signaling in the green algae. These important signaling domains may therefore be widespread among eukaryotes and most probably evolved in ancestral eukaryotes before the divergence of the Opisthokonts (the animal and fungal lineage).

TMPRSS3, a type II transmembrane serine protease mutated in non-syndromic autosomal recessive deafness

Recently, we and others have shown that mutations in TMPRSS3 were responsible for autosomal recessive non-syndromic hearing loss. TMPRSS3 is a member of the Type II Transmembrane Serine Protease (TTSP) family and encodes for a protease that also contains LDLRA (low-density lipoprotein receptor class A) and SRCR (scavenger receptor cysteine rich) domains. Fourteen pathogenic mutations, which occur not only in the catalytic domain but also in the LDLRA and SRCR domains, have been identified to date that cause the DFNB8/10 forms of deafness. In vitro experiments demonstrated that TMPRSS3 mutants were proteolytically inactive indicating that TMPRSS3 protease activity is critical for normal auditory function. However, how missense mutations in the LDLRA and SRCR domains affect the proteolytic activity of TMPRSS3 remains to be elucidated. Although the role of TMPRSS3 in the auditory system is currently not completely understood, it has been shown to regulate the activity of the ENaC sodium channel in vitro and could therefore participate in the regulation of sodium concentration in the cochlea. TMPRSS3 mutations are not a common cause of hereditary deafness, the elucidation of its function is nevertheless important for better understanding of hearing, and provide biological targets for therapeutic interventions.

Salivary agglutinin/glycoprotein-340/DMBT1: a single molecule with variable composition and with different functions in infection, inflammation and cancer

Salivary agglutinin (SAG), lung glycoprotein-340 (gp-340) and Deleted in Malignant Brain Tumours 1 (DMBT1) are three names for identical proteins encoded by the dmbt1 gene. DMBT1/SAG/gp-340 belongs to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins, a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. On the one hand, DMBT1 may represent an innate defence factor acting as a pattern recognition molecule. It interacts with a broad range of pathogens, including cariogenic streptococci and Helicobacter pylori, influenza viruses and HIV, but also with mucosal defence proteins, such as IgA, surfactant proteins and MUC5B. Stimulation of alveolar macrophage migration, suppression of neutrophil oxidative burst and activation of the complement cascade point further to an important role in the regulation of inflammatory responses. On the other hand, DMBT1 has been demonstrated to play a role in epithelial and stem cell differentiation. Inactivation of the gene coding for this protein may lead to disturbed differentiation, possibly resulting in tumour formation. These data strongly point to a role for DMBT1 as a molecule linking innate immune processes with regenerative processes.

Differential Gene Expression in a Marine Sponge in Relation to Its Symbiotic State

The molecular mechanisms involved in the establishment and maintenance of sponge photosymbiosis, and in particular the association with cyanobacteria, are unknown. In the present study we analyzed gene expression in a common Mediterranean sponge (Petrosia ficiformis) in relation to its symbiotic (with cyanobacteria) or aposymbiotic status. A screening approach was applied to identify genes expressed differentially in symbiotic specimens growing in the light and aposymbiotic specimens growing in a dark cave at a short distance from the illuminated specimens. Out of the various differentially expressed sequences, we isolated two novel genes (here named PfSym1 and PfSym2) that were up-regulated when cyanobacterial symbionts were harbored inside the sponge cells. The sequence of one of these genes (PfSym2) was found to contain a conserved domain: the scavenger receptor cysteine rich (SRCR) domain. This is the first report on the expression of sponge genes in relation to symbiosis and, according to the presence of an SRCR domain, we suggest possible functions for one of the genes found in the sponge-cyanobacteria symbiosis.

Gating the Radical Hemoglobin to Macrophages: The Anti-Inflammatory Role of CD163, a Scavenger Receptor

Efficient extracellular hemoglobin (Hb) clearance is essential to prevent oxidative- and nitrosative-mediated toxicity. CD163 belongs to group B of the scavenger receptor cysteine-rich (SRCR) protein family found on the surface of monocytes and macrophages and is responsible for Hb-haptoglobin (Hp) complex uptake. Hb uptake by CD163 was thought to proceed exclusively through an Hp-dependent pathway. However, Hb can interact directly with CD163 via a low affinity binding when Hp is absent. As a result, a two-phase hypothesis of Hb clearance by monocytes/macrophages suggests that Hp-Hb binding to CD163 is the primary mechanism of plasma Hb clearance, while clearance of Hb by direct binding to CD163 is secondary to Hp depletion. The authors have considered the ligand specificity of CD163 in human macrophages and in a heterologous gene expression model to demonstrate that Hb is effectively endocytosed by CD163 in the absence of Hp. Additionally, the authors have considered Hb-based oxygen carriers (HBOCs) administration as a unique situation during which direct CD163 uptake may be relevant as a mechanism of clearance. However, the nature of chemical modifications introduced onto the Hb molecule and/or oxidative changes induced in the protein appear to influence the extent of CD163 interaction and cellular uptake. Here, an overview and novel insights into the role of CD163 in Hb redox inactivation and clearance are provided.

The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor

AbstractErythropoiesis occurs in erythroblastic islands, where developing erythroblasts closely interact with macrophages. The adhesion molecules that govern macrophage-erythroblast contact have only been partially defined. Our previous work has implicated the rat ED2 antigen, which is highly expressed on the surface of macrophages in erythroblastic islands, in erythroblast binding. In particular, the monoclonal antibody ED2 was found to inhibit erythroblast binding to bone marrow macrophages. Here, we identify the ED2 antigen as the rat CD163 surface glycoprotein, a member of the group B scavenger receptor cysteine-rich (SRCR) family that has previously been shown to function as a receptor for hemoglobin-haptoglobin (Hb-Hp) complexes and is believed to contribute to the clearance of free hemoglobin. CD163 transfectants and recombinant protein containing the extracellular domain of CD163 supported the adhesion of erythroblastic cells. Furthermore, we identified a 13–amino acid motif (CD163p2) corresponding to a putative interaction site within the second scavenger receptor domain of CD163 that could mediate erythroblast binding. Finally, CD163p2 promoted erythroid expansion in vitro, suggesting that it enhanced erythroid proliferation and/or survival, but did not affect differentiation. These findings identify CD163 on macrophages as an adhesion receptor for erythroblasts in erythroblastic islands, and suggest a regulatory role for CD163 during erythropoiesis.

Crystal Structure of the Cysteine-rich Domain of Scavenger Receptor MARCO Reveals the Presence of a Basic and an Acidic Cluster That Both Contribute to Ligand Recognition

MARCO is a trimeric class A scavenger receptor of macrophages and dendritic cells that recognizes polyanionic particles and pathogens. The distal, scavenger receptor cysteine-rich (SRCR) domain of the extracellular part of this receptor has been implicated in ligand binding. To provide a structural basis for understanding the ligand-binding mechanisms of MARCO, we have determined the crystal structure of the mouse MARCO SRCR domain. The recombinant SRCR domain purified as monomeric and dimeric forms, and their structures were determined at 1.78 and 1.77 A resolution, respectively. The monomer has a compact globular fold with a twisted five-stranded antiparallel beta-sheet and a long loop covering a single alpha-helix, whereas the dimer is formed via beta-strand swapping of two monomers, thus containing a large eight-stranded beta-sheet. Calculation of the surface electrostatic potential revealed that the beta-sheet region with several arginines forms a basic cluster. Unexpectedly, an acidic cluster was found in the long loop region. In the monomer, the acidic cluster is involved in metal ion binding. Studies with cells expressing various SRCR domain mutants showed that all of the arginines of the basic cluster are involved in ligand binding, suggesting a cooperative binding mechanism. Ligand binding is also dependent on the acidic cluster and Ca2+ ions whose depletion appears to affect ligand binding at least by modulating the electrostatic potential or relative domain orientation. We propose that the SRCR domain dimerization can contribute to the recognition of large ligands by providing a means for the MARCO receptor oligomerization.

Crystal Structure of the Third Extracellular Domain of CD5 Reveals the Fold of a Group B Scavenger Cysteine-rich Receptor Domain

Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among cell surface and secreted proteins of the innate and adaptive immune system, where they mediate ligand binding. We have solved the crystal structure at 2.2 A of resolution of the SRCR CD5 domain III, a human lymphocyte receptor involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. The first structure of a member of a group B SRCR domain reveals the fold of this ancient protein module into a central core formed by two antiparallel beta-sheets and one alpha-helix, illustrating the conserved core at the protein level of genes coding for group A and B members of the SRCR superfamily. The novel SRCR group B structure permits the interpretation of site-directed mutagenesis data on the binding of activated leukocyte cell adhesion molecule (ALCAM/CD166) binding to CD6, a closely related lymphocyte receptor homologue to CD5.

Extracellular Isoforms of CD6 Generated by Alternative Splicing Regulate Targeting of CD6 to the Immunological Synapse

The great majority of mammalian genes yield multiple transcripts arising from differential mRNA processing, but in very few instances have alternative forms been assigned distinct functional properties. We have cloned and characterized a new isoform of the accessory molecule CD6 that lacks the CD166 binding domain and is expressed in rat and human primary cells. The novel isoform, CD6Deltad3, results from exon 5 skipping and consequently lacks the third scavenger receptor cysteine-rich (SRCR) domain of CD6. Differential expression of the SRCR domain 3 resulted in a remarkable functional difference: whereas full-length CD6 targeted to the immunological synapse, CD6Deltad3 was unable to localize at the T cell:APC interface during Ag presentation. Analysis of expression of CD6 variants showed that, while being more frequent in coexpression with full-length CD6, the CD6Deltad3 isoform constituted the sole species in a small percentage of T cells. In the rat thymus, CD6Deltad3 is less represented in double-positive thymocytes but is detectable in nearly 50% of single-positive CD4 or CD8 thymocytes, suggesting that CD6 switching between full-length and Deltad3 isoforms may be involved in thymic selection. Strikingly, CD6Deltad3 is markedly up-regulated upon activation of T lymphocytes, partially substituting full-length CD6, as evaluated by RT-PCR analysis at the single-cell level, by immunoblotting, and by flow cytometry using Abs recognizing SRCR domains 1 and 3 of human CD6. This elegant mechanism controlling the expression of the CD166 binding domain may help regulate signaling delivered by CD6, through different types of extracellular engagement.

Expression, purification and crystallization of human CD5 domain III, a nano-scale crystallization example

The human lymphocyte receptor CD5, a key regulator of immune responses, is involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. CD5 is a membrane glycoprotein which belongs to the group B scavenger receptor cysteine-rich (SRCR) superfamily for which no structural information is available. The most conserved membrane-proximal SRCR domain of CD5 (domain III) has been expressed in HEK-EBNA-293 cells. Although the yield of the purified protein was at the level of micrograms, well diffracting crystals have been obtained. The crystals belong to a tetragonal space group P4 122 or P4 322. They contain two molecules per asymmetric unit and diffracted to 2.5 Å resolution using synchrotron radiation. The strategy shown here to produce, isolate and crystallize CD5 domain III can be used for other mammalian proteins difficult to produce for structural or other biophysical studies.