Accuri C6 Flow Cytometer System
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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0704407

Received for publication July 16, 2004.
Revised December 29, 2004.
Accepted for publication December 30, 2004.

Article

Epitope mapping of Ly-49G and G-like receptors: CK-1 antibody defines a polymorphic site of functional interaction with class I ligand

Mohammed S. Osman , Elizabeth T. Silver , Jay C. Varghese , Chew Shun Chang , Dong-Er Gong , Gerald F. Audette , Bart Hazes , and Kevin P. Kane @

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada

@ To whom correspondence should be addressed. E-mail: kevin.kane{at}ualberta.ca.


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Abstract

Ly-49 receptors regulate mouse natural killer cell functions. Members of the polymorphic Ly-49 multigene family recognize specific alleles of major histocompatibility complex class I (MHC I) or MHC I-like proteins. Previous studies have provided insight into the nature of Ly-49A and -C interaction with their high-affinity MHC I ligands, H-2Dd and Kb, respectively. Unlike Ly-49C, recognition of MHC I by Ly-49A is regulated in part by residues within the {beta}4-{beta}5 loop of its ectodomain. Ly-49A and -G are within the same Ly-49 subfamily, and both receptors recognize Dd. However, there have been no studies that define specific sites on Ly-49G that mediate class I recognition. The Ly-49G receptors of different inbred mouse strains can differ as a result of amino acid polymorphisms within their ectodomains. In this report, we have generated a novel antibody, CK-1, which recognizes Ly-49GB6 and a Ly-49GB6-like receptor, Ly-49Mnonobese diabetic, but not Ly-49GBALB/c. By exploiting differences within ectodomains of C57BL/6 and BALB/c Ly-49G allele products, we identified epitopes recognized by the Ly-49G-specific antibodies CK-1 and Cwy-3, whose epitopes mapped within the {beta}4-{beta}5 loop and the {beta}1 strand, respectively, and were nonoverlapping. Although both antibodies specifically recognized the Ly-49GB6 ectodomain, Cwy-3 was unable to block its interaction with MHC I, and CK-1 significantly inhibited it. The importance of residues within the {beta}4-{beta}5 loop in Ly-49G recognition demonstrates that its interaction with MHC I is similar to that of Ly-49A but not Ly-49C.

Key Words: Ly-49 receptors • motif • ligand interaction