Mapping the CD4 binding domain of gp17, a glycoprotein secreted from seminal vesicles and breast carcinomas.
gp17, a secretory CD4-binding factor isolated from the human seminal plasma, is identical to the gross cystic disease fluid protein-15, a specific marker for primary and metastatic breast tumors. We previously demonstrated that gp17 binds to CD4 with high affinity and strongly inhibits T lymphocyte apoptosis induced by sequential cross-linking of CD4 and T cell receptor (TCR). To further characterize the gp17/CD4 interaction and map the gp17 binding site, we produced a secreted form of recombinant gp17 fused to human IgG1 Fc, gp17-Ig. We showed that gp17-Ig exhibits a binding affinity for CD4 similar to that of native gp17. As no information about gp17 structure is presently available, 99 overlapping gp17 peptides were synthesized by the Spot method, which allowed the mapping of two CD4 binding regions. Alanine scanning of CD4-reactive peptides identified critical residues, selected for site-directed mutagenesis. Nine gp17-Ig mutants were generated and characterized. Three residues within the carboxy-terminal region were identified as the major binding domain to CD4. The Spot method combined with mutagenesis represents a refined approach to distinguish the contact residues from the ones contributing to the conformation of the CD4-binding domain.
Basmaciogullari
S
S
Génétique Moléculaire et Biologie du Développement, ERS 1984, Centre National de la Recherche Scientifique, 19 rue Guy Moquet, 94801 Villejuif, France.
Autiero
M
M
Culerrier
R
R
Mani
J C
JC
Gaubin
M
M
Mishal
Z
Z
Guardiola
J
J
Granier
C
C
Piatier-Tonneau
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001053:Apolipoproteins; D053399:Apolipoproteins D; D001943:Breast Neoplasms / Q000378:metabolism*; D015704:CD4 Antigens / Q000378:metabolism; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D008297:Male; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012669:Seminal Vesicles / Q000378:metabolism*; D014162:Transfection
C024328:APOD protein, human; D001053:Apolipoproteins; D053399:Apolipoproteins D; D015704:CD4 Antigens; D002352:Carrier Proteins; D006023:Glycoproteins; D007074:Immunoglobulin G; D026901:Membrane Transport Proteins; D009363:Neoplasm Proteins; C488892:PIP protein, human; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins