HUMAN KERATINOCYTE GROWTH-FACTOR RECOMBINANTLY EXPRESSED IN CHINESE-HAMSTER OVARY CELLS - ISOLATION OF ISOFORMS AND CHARACTERIZATION OF POSTTRANSLATIONAL MODIFICATIONS

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that acts specifically on epithelial cells in a paracrin e mode. We employed a mammalian expression system to synthesize recomb inant human KGF and isolated two preparations, KGF-a and KGF-b, from m edium conditioned by Chinese hamster ovary cells. On an SDS-PAGE gel, KGF-a migrates as two bands near 25-29 kDa and contains both N- and O- linked sugar moieties attached near the N-terminus. Detailed structura l characterization confirms that KGF-a contains a single amino acid se quence predicted from cDNA sequence and the molecule has two intramole cular disulfide bridges, Cys(1)-Cys(15) and Cys(102)-Cys(106). An addi tional Cys at position 40 is free and resides in a solvent-inaccessibl e environment. Mass spectrometric analyses of HGF-a peptides verify th e occurrence of several post-translational modifications in the molecu le, including partial oxidation at Met(28), partial sulfation at Tyr(2 7), and glycosylation at Asn(14) and Thr(22). Th,Asn-linked carbohydra te structures are heterogeneous, which include biantennary, triantenna ry, and tetraantennary structures with none or up to four sialic acids attached to various structures, while the Thr-linked carbohydrates co ntain typical mucin-type structures. KGF-b is an N-terminally truncate d form of KGF-a posttranslationally processed at Arg(23) and is not gl ycosylated. Both KGF-a and KGF-b forms are capable of stimulating DNA synthesis in quiescent Balb/MK mouse epidermal keratinocytes. (C) 1998 Academic Press.