title: Disulfide structure of the heparin binding domain in vascular endothelial growth factor: characterization of posttranslational modifications in VEGF.
abstract: Preparations of recombinant human vascular endothelial growth factor (VEGF165) expressed in Chinese hamster ovary (CHO) cells and Escherichia coli were compared using a variety of analytical methods. Amino terminal sequence analyses of both the CHO- and E. coli-derived VEGF165 confirmed the predicted amino terminal sequence for VEGF165, although the CHO VEGF165 exhibited a heterogeneous amino terminus with sequences beginning at Ala-1 (76%), Pro-2 (4%), Ala-4 (13%), and Glu-5 (7%). Tryptic digests of reduced and carboxymethylated CHO- and E. coli-derived VEGF165 were examined by LC/MS analyses, indicating equivalent primary structure, except for the glycosylation at Asn-75 in the CHO-derived VEGF165. The N-linked carbohydrate in the CHO-derived VEGF165 was determined to be a complex fucosylated biantennary structure. The data obtained from LC/MS analysis and amino terminal sequence analysis of VEGF165 confirmed 98% of the primary structure. Disulfide linkages for the eight cysteine residues in the carboxyl terminal heparin binding domain were assigned by amino terminal sequencing of fragments produced by tryptic digests of each native molecule. The following disulfides have been identified for both CHO- and E. coli-derived VEGF165: Cys-117 and Cys-135, Cys-120 and Cys-137, Cys-139 and Cys-158, plus Cys-146 and Cys-160. Plasmin cleavage of VEGF165 yields an N-terminal homodimeric VEGF110 and a 55-amino-acid carboxyl terminal domain. VEGF110 was resistant to further proteolytic or chemical digestion such that the disulfide linkages were not elucidated. The 55-amino-acid carboxyl terminal region of VEGF165 appears to be a unique heparin binding domain with no known protein homology.
authors.affiliation: Department of Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: R G
authors.initials: RG
authors.lastname: Keck
authors.forename: L
authors.initials: L
authors.lastname: Berleau
authors.forename: R
authors.initials: R
authors.lastname: Harris
authors.forename: B A
authors.initials: BA
authors.lastname: Keyt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D016228:Endothelial Growth Factors / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008222:Lymphokines / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D004220:Disulfides; D016228:Endothelial Growth Factors; D008222:Lymphokines; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D006493:Heparin; D014357:Trypsin