ABSTRACT
The scavenger receptor cysteine-rich superfamily (SRCR-SF) members are transmembrane and/or secreted receptors exhibiting one or several repeats of a cysteine-rich protein module of â¼100 aa, named scavenger receptor cysteine-rich (SRCR). Two types of SRCR domains (A or B) have been reported, which differ in the number of coding exons and intradomain cysteines. Although no unifying function has been reported for SRCR-SF members, recognition of pathogen-associated molecular patterns (PAMPs) was recently shown for some of them. In this article, we report the structural and functional characterization of mouse S5D-SRCRB, a new group B member of the SRCR-SF. The s5d-srcrb gene maps at mouse chromosome 7 and encompasses 14 exons extending over 15 kb. The longest cDNA sequence found is 4286 bp in length and encodes a mature protein of 1371 aa, with a predicted M(r) of 144.6 kDa. Using an episomal mammalian-expression system, a glycosylated soluble recombinant form >200 kDa was obtained and used as immunogen for the generation of specific rat mAbs. Subsequent immunohistochemical and real-time PCR analysis showed significant S5D-SRCRB expression in murine genitourinary and digestive tracts. S5D-SRCRB was shown to bind endogenous extracellular matrix proteins (laminin and galectin-1), as well as PAMPs present on Gram-positive and Gram-negative bacteria and fungi. PAMP binding by S5D-SRCRB induced microbial aggregation and subsequent inhibition of PAMP-induced cytokine release. These abilities suggest that S5D-SRCRB might play a role in the innate defense and homeostasis of certain specialized epithelial surfaces.
Subject(s)
Cysteine/metabolism , Gene Expression Regulation/immunology , Multigene Family/immunology , Scavenger Receptors, Class B/chemistry , Scavenger Receptors, Class B/physiology , Amino Acid Sequence , Animals , Epithelial Cells/chemistry , Epithelial Cells/metabolism , Epithelial Cells/physiology , HEK293 Cells , Homeostasis/genetics , Homeostasis/immunology , Humans , Immunity, Innate/genetics , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Protein Binding/immunology , Protein Structure, Tertiary/genetics , Rats , Rats, Sprague-Dawley , Receptors, Pattern Recognition/chemistry , Receptors, Pattern Recognition/metabolism , Receptors, Pattern Recognition/physiology , Scavenger Receptors, Class B/biosynthesis , Scavenger Receptors, Class B/geneticsABSTRACT
Human Sp alpha is a soluble protein belonging to group B of the scavenger receptor cysteine-rich (SRCR) superfamily for which little functional information is available. It is expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), and it binds to myelomonocytic and lymphoid cells, which suggests that it may play an important role in the regulation of the innate and adaptive immune systems. In the present study we show that recombinant human Sp alpha (rSp alpha) binds to the surface of several gram-positive and gram-negative bacterial strains. Competition studies indicated that such binding is mediated by the recognition of lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, through nonoverlapping sites on the Sp alpha molecule. The most conserved part of LPS (2-keto-3-deoxyoctulosonic acid and lipid A) was shown to be involved in the recognition by Sp alpha. Bacterial binding studies using the SRCR domain 1 of Sp alpha showed that this domain retains both the LPS and LTA binding activities, indicating that both bacterial interacting sites are retained in a single SRCR domain. Furthermore, rSp alpha induced aggregation of gram-positive and gram-negative bacteria strains. On the other hand, rSp alpha inhibited tumor necrosis factor-alpha secretion by human monocytes stimulated with LPS or LTA. Binding of Sp alpha to conserved components of bacterial surfaces and modulation of the monocyte response indicate that this molecule is an active constituent of the innate immune response of the host.
Subject(s)
Receptors, Immunologic/physiology , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins , Bacteria/metabolism , Binding Sites , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Escherichia coli/metabolism , Humans , Immunity, Innate , Lipopolysaccharides/chemistry , Lipopolysaccharides/pharmacology , Listeria/metabolism , Macrophages/metabolism , Mice , Molecular Sequence Data , Monocytes/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, Immunologic/chemistry , Receptors, Scavenger/chemistry , Recombinant Proteins/chemistry , Scavenger Receptors, Class B , Sequence Homology, Amino Acid , Spectrometry, Fluorescence , Teichoic Acids/chemistry , Temperature , Tissue Distribution , Tumor Necrosis Factor-alpha/metabolismABSTRACT
CD6 is a type I membrane glycoprotein expressed on thymocytes, mature T and B1a lymphocytes, and CNS cells. CD6 binds to activated leukocyte cell adhesion molecule (CD166), and is considered as a costimulatory molecule involved in lymphocyte activation and thymocyte development. Accordingly, CD6 partially associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse (IS) on T lymphocytes. However, the signaling pathway used by CD6 is still mostly unknown. The yeast two-hybrid system has allowed us the identification of syntenin-1 as an interacting protein with the cytoplasmic tail of CD6. Syntenin-1 is a PDZ (postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1) domain-containing protein, which functions as an adaptor protein able to bind cytoskeletal proteins and signal transduction effectors. Mutational analyses showed that certain amino acids of the most C-terminal sequence of CD6 (-YDDISAA) and the two postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1 domains of syntenin-1 are relevant to the interaction. Further confirmation of the CD6-syntenin-1 interaction was obtained from pull-down and co-immunoprecipitation assays in mammalian cells. Image analyses also showed that syntenin-1 accumulates at CD6 caps and at the IS. Therefore, we propose that syntenin-1 may function as a scaffolding protein coupling CD6 and most likely other lymphocyte receptors to cytoskeleton and/or signaling effectors during IS maturation.
