Disulfide structure of the heparin binding domain in vascular endothelial growth factor: Characterization of posttranslational modifications in VEGF

Preparations of recombinant human vascular endothelial growth factor (VEGF(165)) 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 VEGF(165) confirmed the predicted amino terminal sequence for VEGF(165) although the CHO VEGF(165) 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 VEGF(165) were examined by LC/MS analyses, indicating equivalent primary structure, except for the glycosylation at Asn-75 in the CHO-derived VEGF(165). The N-linked carbohydrate in the CHO-derived VEGF(165) was determined to be a complex fucosylated biantennary structure, The data obtained from LC/MS analysis and amino terminal sequence analysis of VEGF(165) 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 VEGF(165): Cys-117 and Cys-135, Cys-120 and Cys-137, Cys-139 and Cys-158, plus Cys-146 and Cys-160, Plasmin cleavage of VEGF(165) yields an N-terminal homodimeric VEGF(110) and a 55-amino-acid carboxyl terminal domain, VEGF(110) was resistant to further proteolytic or chemical digestion such that the disulfide linkages were not elucidated, The 55-amino-acid carboxyl terminal region of VEGF(165) appears to be a unique heparin binding domain with no known protein homology. (C) 1997 Academic Press.