Olga Padilla

Interaction of recombinant and natural soluble CD5 forms with an alternative cell surface ligand

CD5, a member of the scavenger receptor cysteine‐rich (SRCR) receptor family, plays a role in the thymocyte maturation, T cell activation and T cell‐antigen‐presenting cell interactions. To date only CD5 ligands (CD5L) compatible with a T‐B co‐stimulatory role have been described (CD72, gp40‐80 and IgVH framework region) so the existence of alternative CD5L involved in other aspects of T cell biology warrants further exploration. Here we characterize the cell binding properties of a recombinant soluble human CD5 extracellular domain glycoprotein (rsCD5). In contrast to previously characterized ligands, this molecule binds to a broadly distributed cell surface receptor expressed on monocytes, lymphocytes and various cell lines of lymphoid, myelomonocytic and epithelial origin. The cell binding of rsCD5 is divalent cation independent and inhibited by high molar concentrations of certain monosaccharides. Both human CD5 Ig fusion proteins and a natural soluble CD5 form (present in human serum and resulting from proteolytic cleavage following lymphocyte activation) reproduce the cell binding pattern of rsCD5 and block its binding in a competitive form. The involvement of the most N‐terminal CD5 SRCR domains (D1 and D2) in binding is deduced from competition cell binding assays with CD5 Ig fusion proteins. These results imply a novel CD5/CD5L interaction model recalling some aspects of the interaction of CD6 with activated leukocyte cell adhesion molecule (ALCAM).

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 Å 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 β-sheets and one α-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.

Residues Y429 and Y463 of the human CD5 are targeted by protein tyrosine kinases

The human CD5 lymphocyte cell surface co‐receptor modulates activation and differentiation responses mediated by the antigen‐specific receptor of T and B cells. CD5 is phosphorylated followinglymphocyte activation; however, the exact sites and kinases involved are yet to be determined. Jurkat T cell transfectants expressing tyrosine‐mutated CD5 molecules have been used to show that residues Y429 and Y463 are targeted in vivo by protein tyrosine kinases following cell stimulation with anti‐CD3 mAb or pervanadate. This is in agreement with data from direct in vitrokinase assays using purified recombinant Lck and Fyn protein tyrosine kinases. The analysis of Lck‐ and CD3‐deficient Jurkat cells shows that tyrosine phosphorylation of CD5 requires Lck activity. We propose that T cell activation mediates CD5 tyrosine phosphorylation at residues Y429 and Y463 mainly through the activation of Lck.

Role of Two Conserved Cytoplasmic Threonine Residues (T410 and T412) in CD5 Signaling

CD5 is a transmembrane coreceptor that modulates activation and differentiation signals mediated by the Ag-specific receptor present on both T and B1a lymphocytes. CD5 lacks intrinsic catalytic activity, and its immunomodulatory properties result from intracellular interactions mediated by the CD5 cytoplasmic tail. The nature of these interactions is currently a matter of investigation. Here, we present a selective mutagenesis analysis of two conserved threonine residues (T410 and T412) located at the membrane-proximal cytoplasmic region of CD5. These residues are contained within consensus phosphorylation motifs for protein kinase C and are shown here to be critical for in vivo protein kinase C-mediated phosphorylation of CD5. Functional studies revealed that the integrity of T410 and T412 is also critical for CD5-mediated phosphatidylcholine-specific phospholipase C (PC-PLC) activation and phorbol ester-mediated inhibition of Ab-induced internalization of CD5. These results strongly argue in favor of a role for T410 and T412 in the signaling mediated by CD5.

Molecular and Functional Characterization of Mouse S5D-SRCRB: A New Group B Member of the Scavenger Receptor Cysteine-Rich Superfamily

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 ∼100 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 Mr 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.