Purification and characterization of 33.5 kDa vesicular protein in human bile.
OBJECTIVE
The present study was undertaken to purify and partially characterize the 33.5-kilodalton (33.5 kDa) vesicular protein in human bile and to explore the possible molecular mechanisms of the initial crystal nucleation process.


METHODS
The 33.5 kDa vesicular protein was isolated by ultracentrifugation and further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. The purified 33.5 kDa vesicular protein was subjected to N-terminal amino acid sequencing and amino acid analysis. Cholesterol crystallization activity was detected by cholesterol crystal growth assay. The sugar chain of the 33.5 kDa vesicular protein was analyzed by dot-immunobinding assay of lectin coupled to a peroxidase (HRP-DSA, HRP-ConA, HRP-WGA) and was deglycosylated using two different enzymatic approaches (N-deglycosylation and O-deglycosylation) to determine the molecular weight of the protein component, the type of linkage between polypeptide and carbohydrate components.


RESULTS
The 33.5 kDa vesicular protein with complicated glycan was an extensively glycosylated (37.3%) monomer and these sugar chains strongly bound to DSA, but did not bind to ConA. Amino acid sequencing indicated that the protein was unique. The 33.5 kDa vesicular protein exhibited potent cholesterol crystallization promoting activity in vitro with derived crystal growth curve indices It, Ig, Ic presented as 0.57, 1.52, and 1.63 respectively. Both enzymatic proteolysis and N-deglycosylation of the protein removed all activity.


CONCLUSIONS
These data suggest the 33.5 kDa vesicular protein may be responsible for the pathogenesis of cholesterol gallstone disease, and the sugar chains play an important role in pro-nucleating process.
Xiang
Jian-Bin
JB
Department of General Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China. xjbzhw@yahoo.com.cn
Cai
Duan
D
Ma
Bao-Jin
BJ
Cha
Xi-Liang
XL
Wang
Li-Ying
LY
Mo
Han-Qing
HQ
Zhang
Yan-Ling
YL
D001646:Bile / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry; D003460:Crystallization; D042882:Gallstones / Q000737:chemistry*; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D014461:Ultracentrifugation
D006023:Glycoproteins; D002784:Cholesterol