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© The Rockefeller University Press, 0022-1007/1999/8/331/ $5.00
The Journal of Experimental Medicine, Volume 190, Number 3, August 2, 1999 331-340

Original Article

Homologue Scanning Mutagenesis Reveals Cd66 Receptor Residues Required for Neisserial Opa Protein Binding

Martine P. Bosa, Daniel Hogana, and Robert J. Bellanda

a From the Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840-2999
Rocky Mountain Laboratories, NIH, NIAID, 903 South 4th St., Hamilton, MT 59840.406-363-9204406-363-9301

Martine_Bos{at}nih.gov

The immunoglobulin-like family of CD66 antigens, present on human neutrophils and epithelial cells, are used as receptors for adhesins expressed by the pathogenic Neisseriae. N. gonorrhoeae strain MS11 can express 11 isoforms of these adhesins, called opacity-related (Opa) proteins. Each MS11 Opa protein recognizes a distinct spectrum of CD66 receptors. CD66–Opa binding is mediated by the NH2-terminal domain of the receptor and occurs through protein–protein interactions. In this report, we have investigated the molecular basis for the binding between the CD66 and Opa protein families by mapping amino acids in CD66 receptors that determine Opa protein binding. We performed homologue scanning mutagenesis between CD66e, which binds multiple Opa variants, and CD66b, which binds none, and tested both loss-of-function by CD66e and gain-of-function by CD66b in solution assays and in assays involving full-length receptors expressed by epithelial cells. We found that three residues in the CD66e N-domain are required for maximal Opa protein receptor activity. Opa proteins that recognize the same spectrum of native CD66 molecules showed differential binding of receptors with submaximal activity, indicating that the binding characteristics of these Opa proteins are actually slightly different. These data provide a first step toward resolving the structural requirements for Opa–CD66 interaction.

Key Words: Neisseria gonorrhoeae • carcinoembryonic antigen • bacterial adhesion • opacity protein • mutagenesis

1used in this paper: CAM, cell adhesion molecule; CHO, Chinese hamster ovary; CEA, carcinoembryonic antigen; GAM, goat anti–mouse; GAR, goat anti–rabbit; ICAM, intercellular adhesion molecule; IgSF, immunoglobulin superfamily; mut, mutant; N-domain, NH2-terminal Ig variable–like domain; Ngo, Neisseria gonorrhoeae; Nme, Neisseria meningitidis; Opa, opacity-related

© 1999 The Rockefeller University Press


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