STUDY OF AN ANTI-HCG MONOCLONAL ANTIBODY COMPLEXED WITH ITS PEPTIDE ANTIGEN

Constantina Fotinou1, Isobel Black1, Paul Emsley1, Neil W. Isaacs1, Annemarie deHaan2, Ebo Bos2 , Wim J.G. Schielen3

1Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK
2Dept. of Biotechnology & Biochemistry, N.V. Organon, Molenstraat 110, PO Box 20, 5340 BH, Oss, The Netherlands.
3Chemistry Research Unit, Organon Teknika BV,Box 84, 5280 AB Boxtel, The Netherlands

Human chorionic gonadotropin (hCG) is a glycoprotein hormone essential for the maintenance of pregnancy [1] [2]. Apart from its physiological action, hCG is found in pathological cases such as choriocarcinoma and testicular cancer [3] Holo-hCG, its free subunits, or nicked parts of them are secreted during the course of these diseases and thus can be used as immunological markers of these conditions [4].

The family of heterodimeric glycoprotein hormones comprises chorionic gonadotropin, luteinising hormone (LH), follicle-stimulating hormone (FSH) and thyroid stimulating hormone (TSH). Within a given species the shorter a-subunit is common to the four hormones and is immunologically indistinguishable [2]. The b-subunits are distinctive for each hormone and confer specific biological and immunological activity. Both subunits have two N-linked glycosylation sites. The b-subunit of hCG has a unique 31 residue (residues 115-145) C-terminal peptide containing four O-linked glycosylation sites. Antibodies against this peptide have been used as experimental contraception vaccines [5].

We present here the structure of an Fab fragment of a monoclonal antibody (Fab3A2) against an epitope on the C-terminal region of the b-subunit of hCG complexed with a synthetic peptide homolog to the b132-145 residues.

The strucure has been determined to 2.3 A resolution. The crystals are very thin plates, with dimensions of 0.4x0.4x0.02mm and diffract to 1.8 A. Data were collected in SRS on station 9.6, under cryo-cooling conditions (100K). The structure was solved by Molecular Replacement using AMORE [6] with the uncomplexed Fab3A2 [7] as a search model. Refinement of the structure was performed using Refmac [8] and ARP [9]. The crystallographic R factor is currently 23.1\% and the Rfree is 30.4\%. 100 water molecules are included in the current model.

Structure description

The structure reveals a typical immunoglobulin fold for the Fab3A2 fragment. A comparison of the structure of the bound to the peptide Fab3A2 with the uncomplexed one shows a peptide-induced relocation of the H3-loop of the antigen binding site and a rotation of the side chain of the L50 residue (Kabat sequence).

The peptide adopts an extended conformation. There are hydrogen bonds between the residues of the L1 and L2 loops of the antigen binding site and residues of the peptide.

 

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