The Top of the Inserted-like Domain of the Integrin Lymphocyte Function-associated Antigen-1 β Subunit Contacts the α Subunit β-Propeller Domain near β-Sheet 3

Qun Zang,Chafen Lu,Chichi Huang,Junichi Takagi,Timothy A Springer

doi:10.1074/jbc.m002883200

Abstract

We find that monoclonal antibody YTA-1 recognizes an epitope formed by a combination of the integrin alpha(L) and beta(2) subunits of LFA-1. Using human/mouse chimeras of the alpha(L) and beta(2) subunits, we determined that YTA-1 binds to the predicted inserted (I)-like domain of the beta(2) subunit and the predicted beta-propeller domain of the alpha(L) subunit. Substitution into mouse LFA-1 of human residues Ser(302) and Arg(303) of the beta(2) subunit and Pro(78), Thr(79), Asp(80), Ile(365), and Asn(367) of the alpha(L) subunit is sufficient to completely reconstitute YTA-1 reactivity. Antibodies that bind to epitopes that are nearby in models of the I-like and beta-propeller domains compete with YTA-1 monoclonal antibody for binding. The predicted beta-propeller domain of integrin alpha subunits contains seven beta-sheets arranged like blades of a propeller around a pseudosymmetry axis. The antigenic residues cluster on the bottom of this domain in the 1-2 loop of blade 2, and on the side of the domain in beta-strand 4 of blade 3. The I domain is inserted between these blades on the top of the beta-propeller domain. The antigenic residues in the beta subunit localize to the top of the I-like domain near the putative Mg(2+) ion binding site. Thus, the I-like domain contacts the bottom or side of the beta-propeller domain near beta-sheets 2 and 3. YTA-1 preferentially reacts with activated LFA-1 and is a function-blocking antibody, suggesting that conformational movements occur near the interface it defines between the LFA-1 alpha and beta subunits.

Highlights

Lymphocyte function-associated antigen-1 (LFA-1)1 is a member of the leukocyte integrin family: LFA-1 (␣L␤2; CD11a/ CD18), Mac-1 (␣M␤2; CD11b/CD18), p150,95 (␣X␤2; CD11c/ CD18), and ␣D␤2 [1, 2]
We find that monoclonal antibody YTA-1 recognizes an epitope formed by a combination of the integrin ␣L and ␤2 subunits of LFA-1
Monoclonal Antibody YTA-1 Recognizes Human LFA-1 on an Epitope Formed by a Combination of the ␣L and ␤2 Subunits—To test whether the YTA-1 mouse anti-human antibody was specific for both the human ␣L and ␤2 subunits, they were expressed in association with murine ␤2 and ␣L subunits, respectively

Summary

Introduction

Lymphocyte function-associated antigen-1 (LFA-1) is a member of the leukocyte integrin family: LFA-1 (␣L␤2; CD11a/ CD18), Mac-1 (␣M␤2; CD11b/CD18), p150,95 (␣X␤2; CD11c/ CD18), and ␣D␤2 [1, 2]. Extensive studies including mutagenesis and mapping epitopes of function- blocking or activating antibodies have demonstrated that the I domain and the ␤-propeller domain of the ␣L subunit and the I-like domain of the ␤2 subunit cooperatively contribute to ligand binding for LFA-1 (26 –30). We demonstrate that YTA-1 recognizes an activationdependent epitope on LFA-1 consisting of residues from both the ␤2 subunit and the ␣L subunit We identify these specific amino acid residues and their positions in models of the ␣L ␤-propeller domain and the ␤2 I-like domain. Direct association between these subunits is demonstrated and localized

Methods

Results

Conclusion