Determination of Primary Amino Acid Sequence and Unique Three-Dimensional Structure of WGH1, a Monoclonal Human IgM Antibody with Anti-PR3 Specificity 1 Jacquelyn A. Davis, Elisabeth Peen, Ralph C. Williams, Jr., Shane Perkins, Christine C. Malone, Wayne T. McCormack,* Elena Csernok,† W. L. Gross,† A. S. Kolaskar,‡ and Urmila Kulkarni-Kale‡ Department of Medicine and *Department of Pathology, Immunology and Laboratory Medicine, Division of Rheumatology and Clinical Immunology, University of Florida, Gainesville, Florida 32610; †University of Lu ¨ beck, Germany; and ‡Bioinformatics Centre, University of Pune, Ganeshkhind, Pune - 411007, India Transformed B cells making monoclonal IgM- anti- PR3 antibody WGH1 from a patient with Wegener’s granulomatosis were used to prepare mRNA and synthesize cDNA. PCR primers for human  and  chains were then employed to amplify heavy- and light-chain V-regions followed by cloning into pCR2-1 vector and sequencing. Molecular modeling of VH re- gions employed knowledge-based homology modeling to obtain minimum energy conformation. The VH se- quence was subgroup III with marked overall homol- ogy to VH1.9III. The VHCDR3 region of WGH1 was unique, consisting of 21 amino acid residues which included seven tyrosines as well as three negatively charged aspartic acid residues. The VL region was subgroup II with a negatively charged glutamic acid at position 100 in CDR3. Molecular modeling of VH revealed a major conformational difference in the shape of CDR3 compared with other antibodies for which three-dimensional structures have been deter- mined. Monoclonal antibody WGH1 reacting with PR3 (a highly positively charged molecule) shows a unique reactive cassette within VHCDR3 with a number of negatively charged aspartic acid residues. WGH1 VH- CDR3 contains a loop which shows a major projection not usually recorded in other previously studied anti- body molecules. © 1998 Academic Press INTRODUCTION Wegener’s granulomatosis (WG) is a systemic dis- ease of unknown etiology characterized by necrotizing granulomas and vasculitis affecting the upper and lower respiratory tract and kidneys (1, 2). In most WG patients, presence of anti-neutrophil cytoplasmic anti- bodies (C-ANCA) provides a useful diagnostic serologic marker, often paralleling disease activity and tissue inflammatory reaction (3–5). Anti-neutrophil cyto- plasmic antibodies react with a limited spectrum of neutrophil cytoplasmic antigenic materials, including proteinase-3 (PR-3) and in some instances myeloperox- idase or lactoferrin (6 –9). Anti-PR-3 C-ANCA have been studied by a number of investigators, particularly for evidence as to whether they may participate di- rectly in disease pathogenesis. Several possible mech- anisms whereby antibodies with anti-PR-3 specificity might amplify or incite some of the tissue damage or inflammatory process in WG have been suggested (10 – 12), but there is very little immunohistochemical evi- dence for in vivo deposition of anti-PR-3 IgG or other antibodies reacting with neutrophil cytoplasmic com- ponents within pulmonary or renal lesions of WG or polyarteritis patients. PR-3 remains one of the most prominent neutrophil cytoplasmic antigenic constituents reacting with anti- C-ANCA antibodies from patients with active Wegen- er’s involvement. Previous studies have focussed on attempts to define the predominant antigenic epitopes on PR3 (13) or the structural features of V-regions of antibodies reacting with PR-3 or other related neutro- philic cytoplasmic antigens (14). In the present com- munication, we report the primary V-region sequence of a monoclonal human IgM anti-PR3 antibody derived from a cell line (WGH1) produced from a patient with WG and present molecular modeling constructs which show an unusual conformation within the heavy-chain V-region CDR3 of this human monoclonal antibody. MATERIALS AND METHODS Cell culture. Epstein–Barr virus–transformed B cells (WGH1) synthesizing IgM anti-PR-3 isolated from a patient with WG were prepared by E. Csernok in Lu ¨ beck, Germany, as previously described (15). The monoclonal IgM WGH1 showed strong ELISA reactiv- 1 Supported by a grant from the Florida Chapter of the Arthritis Foundation and in part by the Marcia Whitney Schott Endowment to the University of Florida for the Study of Rheumatic Disease. Dr. Peen was supported from the Norwegian Council of Research, Project No. 111159/320. CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Vol. 89, No. 1, October, pp. 35– 43, 1998 Article No. II984582 0090-1229/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. 35