Complete enzymatic deglycosylation of native sex steroid-binding protein (SBP or SHBG) of human and rabbit plasma: effect on the steroid-binding activity.
An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex steroid-binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the steroid-binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of steroid-binding activity and an increase in the Kd for the binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
Petra
P H
PH
Department of Obstetrics & Gynecology, University of Washington, Seattle 98195.
Griffin
P R
PR
Yates
J R
JR
Moore
K
K
Zhang
W
W
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D013196:Dihydrotestosterone / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism; D005260:Female; D006031:Glycosylation; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D011247:Pregnancy; D011374:Progesterone / Q000378:metabolism; D011817:Rabbits; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism
D009844:Oligosaccharides; D012738:Sex Hormone-Binding Globulin; D013196:Dihydrotestosterone; D013739:Testosterone; D011374:Progesterone; D004958:Estradiol; D006854:Hydrocortisone