Deglycosylation of a native, protease-sensitive glycoprotein by peptide N-glycosidase F without protease inhibitors.
The glycoprotein fibrinogen was deglycosylated in its native state and in the absence of protease inhibitors by peptide N-glycosidase F following removal of protease contaminants from the enzyme by HPLC. Fibrinogen is sensitive both to proteolysis by contaminants which may constitute as little as 0.2% of the enzyme protein and to denaturation by 1,10-o-phenanthroline, the only substance known to inhibit the proteolysis. Thus removal of protease contaminants from the enzyme is a prerequisite for the deglycosylation of native fibrinogen. The starting material for the present method is the final material obtained from the purification described by A. L. Tarentino, C. M. Gomez, and T. H. Plummer (1985, Biochemistry 24, 4565). Three sequential passages over a PolyCAT A (20 X .46 cm) cation-exchange column and elutions with a linear gradient of NaCl from 0 to 0.4 M were necessary to completely overcome the tenacious but noncovalent association of peptide N-glycosidase F with contaminants that proteolyze fibrinogen. All three chromatographic runs could be completed in 1 day. Using this "protease-free" enzyme at up to a 1:20 molar ratio, fibrinogen that is completely deglycosylated and native has been generated in order to determine the role of the carbohydrate moieties in its function.
Langer
B G
BG
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Hong
S K
SK
Schmelzer
C H
CH
Bell
W R
WR
D000581:Amidohydrolases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D004340:Drug Contamination; D005340:Fibrinogen / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000494:pharmacology*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011480:Protease Inhibitors / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects
D006023:Glycoproteins; D011480:Protease Inhibitors; D005340:Fibrinogen; D010447:Peptide Hydrolases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase