Naturally Occurring Proteolytic Antibodies: SELECTIVE IMMUNOGLOBULIN M-CATALYZED HYDROLYSIS OF HIV gp120

Sudhir Paul,Sangeeta Karle,Stephanie Planque,Hiroaki Taguchi,Maria Salas,Yasuhiro Nishiyama,Beverly Handy,Robert Hunter,Allen Edmundson,Carl Hanson

doi:10.1074/jbc.m406719200

Abstract

We report the selective catalytic cleavage of the HIV coat protein gp120, a B cell superantigen, by IgM antibodies (Abs) from uninfected humans and mice that had not been previously exposed to gp120. The rate of IgM-catalyzed gp120 cleavage was greater than of other polypeptide substrates, including the bacterial superantigen protein A. The kinetic parameters of gp120 cleavage varied over a broad range depending on the source of the IgMs, and turnover numbers as great as 2.1/min were observed, suggesting that different Abs possess distinct gp120 recognition properties. IgG Abs failed to cleave gp120 detectably. The Fab fragment of a monoclonal IgM cleaved gp120, suggesting that the catalytic activity belongs to the antibody combining site. The electrophoretic profile of gp120 incubated with a monoclonal human IgM suggested hydrolysis at several sites. One of the cleavage sites was identified as the Lys(432)-Ala(433) peptide bond, located within the region thought to be the Ab-recognizable superantigenic determinant. A covalently reactive peptide analog (CRA) corresponding to gp120 residues 421-431 with a C-terminal amidino phosphonate diester mimetic of the Lys(432)-Ala(433) bond was employed to probe IgM nucleophilic reactivity. The peptidyl CRA inhibited the IgM-catalyzed cleavage of gp120 and formed covalent IgM adducts at levels exceeding a control hapten CRA devoid of the peptide sequence. These observations suggest that IgMs can selectively cleave gp120 by a nucleophilic mechanism and raise the possibility of their role as defense enzymes.

Highlights

We report the selective catalytic cleavage of the HIV coat protein gp120, a B cell superantigen, by IgM antibodies (Abs) from uninfected humans and mice that had not been previously exposed to gp120
IgG Abs produced by differentiated B cells, in comparison, contain V domains that are more diversified by adaptive maturational processes occurring after exposure to the antigen
In the case of specific IgG Abs directed to individual antigens, noncovalent Ab-antigen interactions guide the nucleophilic reaction and enable antigen-specific proteolysis, as judged from the selective covalent reaction of the Abs with polypeptide covalently reactive peptide analog (CRA) containing the phosphonate group within their antigenic epitopes (4)

Summary

The abbreviations used are

Antibody; AMC, 7-amino-4-methylcoumarin; BSA, bovine serum albumin; Bt-, biotinylated; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CRA, covalently reactive analog; ELISA, enzyme-linked immunosorbent assay; Fab, fragment antigen binding; FPLC, fast protein liquid chromatography; HIV-1, human immunodeficiency virus type 1; I-protein A, iodinated protein A; sCD4, soluble CD4; sEGFR, soluble epidermal growth factor receptor; V domain, variable domain; VH domain, heavy chain variable domain. We report here the selective and efficient cleavage of the HIV coat protein gp120 by IgM Abs from uninfected humans and mice immunized with irrelevant antigens. Based on their reactivity with CRA probes, the IgMs appear to cleave gp120 via a nucleophilic mechanism. Selectivity for gp120 appears to be derived at least in part from recognition of a peptide determinant composed of gp120 residues 421– 433, which is known to contribute contact sites for CD4 (13, 14) This determinant is implicated in gp120 recognition as a superantigen by the conserved V domain regions of certain Abs (15, 16)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION