Structural requirements for adhesion of soluble recombinant murine vascular cell adhesion molecule-1 to alpha 4 beta 1.
This study describes the identification of seven amino acid residues of the vascular cell adhesion molecule (VCAM-1) that influence binding to the alpha 4 beta 1 receptor. Using recombinant murine VCAM-1-IgG, which is bound by both mouse (WEHI 231) and human (Ramos) lymphoid cells, two approaches demonstrated the crucial role of the first two NH2-terminal Ig-like domains in binding: (a) blocking monoclonal anti-mouse VCAM-1 antibodies bound to only truncation variants that included the first two domains; (b) site-direct mutagenesis of the first NH2-terminal domain showed that alanine substitution of the amino acid residues R36, D40, K46, S54, N65, T72, and E81 partially or completely reduced adherence by human and/or mouse cells. Of these D40, when mutated to A, N, or K (but not E), showed complete abrogation of adherence by mouse and human cells, as well as inability to bind blocking anti-murine VCAM-1 antibody MVCAM.A429, while not inducing gross structural perturbations in VCAM-1. By molecular modeling, the D40 residue was located on a beta turn connecting two beta strands defined as C and D. The residues R36, K46, S54, N65, T72, and E81, which perturb cell adherence and caused small changes to gross structure, are conformationally near or adjacent to D40. Although these residues, identified as crucial for cell adhesion, are all located in domain 1, it is evident that there is a structural requirement for domains 1 and 2 to be intact so that cell adhesive function can occur.
Renz
M E
ME
Department of Immunology, Genentech, Inc., South San Francisco, California 94080.
Chiu
H H
HH
Jones
S
S
Fox
J
J
Kim
K J
KJ
Presta
L G
LG
Fong
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D000939:Epitopes; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D039041:Integrin alpha4beta1; D016023:Integrins / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011947:Receptors, Antigen, B-Cell / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016029:Receptors, Very Late Antigen / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D019010:Vascular Cell Adhesion Molecule-1
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D000939:Epitopes; D007074:Immunoglobulin G; D039041:Integrin alpha4beta1; D016023:Integrins; D011947:Receptors, Antigen, B-Cell; D016029:Receptors, Very Late Antigen; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1