Human keratinocyte growth factor recombinantly expressed in Chinese hamster ovary cells: isolation of isoforms and characterization of post-translational modifications.
Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that acts specifically on epithelial cells in a paracrine mode. We employed a mammalian expression system to synthesize recombinant human KGF and isolated two preparations, KGF-a and KGF-b, from medium 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 structural characterization confirms that KGF-a contains a single amino acid sequence predicted from cDNA sequence and the molecule has two intramolecular disulfide bridges, Cys1-Cys15 and Cys102-Cys106. An additional Cys at position 40 is free and resides in a solvent-inaccessible environment. Mass spectrometric analyses of KGF-a peptides verify the occurrence of several post-translational modifications in the molecule, including partial oxidation at Met28, partial sulfation at Tyr27, and glycosylation at Asn14 and Thr22. The Asn-linked carbohydrate structures are heterogeneous, which include biantennary, triantennary, and tetraantennary structures with none or up to four sialic acids attached to various structures, while the Thr-linked carbohydrates contain typical mucin-type structures. KGF-b is an N-terminally truncated form of KGF-a posttranslationally processed at Arg23 and is not glycosylated. Both KGF-a and KGF-b forms are capable of stimulating DNA synthesis in quiescent Balb/MK mouse epidermal keratinocytes.
Hsu
Y R
YR
Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1890, USA.
Hsu
E W
EW
Katta
V
V
Brankow
D
D
Tseng
J
J
Hu
S
S
Morris
C F
CF
Kenney
W C
WC
Lu
H S
HS
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D051526:Fibroblast Growth Factor 10; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006133:Growth Substances / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D013058:Mass Spectrometry; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism
D017931:DNA Primers; C496453:FGF7 protein, human; C496454:Fgf7 protein, mouse; D051526:Fibroblast Growth Factor 10; D006023:Glycoproteins; D006133:Growth Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase