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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187

Division of Gastroenterology and Hepatology, Departments of ¹Medicine, and ³Biochemistry, Medical College of Wisconsin and Veterans Administration Medical Center, Milwaukee, Wisconsin 53295; and ²Washington University School of Medicine, St. Louis, Missouri 63110

Submitted 24 October 2002 ; accepted in final form 17 March 2003

The current studies have investigated the role of three disulfide bonds of human transcobalamin II (TC II), a plasma transporter of cobalamin (Cbl; vitamin B₁₂), in its function and stability. When translated in vitro in the presence or absence of microsomal vesicles, TC II constructs with a single substitution, C3S or C249S, demonstrated synthesis of a stable functional protein. However, TC II synthesized in the presence of microsomal vesicles using constructs with a single (C98S, C147S, C187S, C291S), double (C3/147/S, C98/147/S) or triple (C3/98/147/S) substitution was unstable. In the absence of microsomal vesicles, the percentage of binding to Cbl-Sepharose matrix by TC II expressed by constructs C3S, C3/147/S, C98/147/S, or C3/98/147/S was 100, 49, 52, and 35%, respectively. Upon their reductive alkylation, the binding of TC II expressed by these constructs was reduced to 25–30%. TC II constructs C3S or C249S, when expressed in TC II-deficient fibroblasts, produced a stable functional protein, but those expressed by constructs C147S, C187S, C291S, C3/147/S, C98/147/S, or C3/98/147/S were rapidly degraded. The intracellular degradation of TC II expressed by these constructs was inhibited by lactacystin or MG-132 but not by the lysosomal degradation inhibitors ammonium chloride or chloroquine. These studies suggest that optimal binding of Cbl by human TC II is supported by disulfide bonds C98-C291 and C147-C187 and that their disruption results in loss of Cbl binding and their rapid degradation by the proteasomal machinery.

secretion; intracellular stability; vitamin B₁₂ binding; proteasome

This article has been cited by other articles:

				S. Kalra, S. Seetharam, R. R. Yammani, and B. Seetharam Rat transcobalamin: cloning and regulation of mRNA expression J. Physiol., April 15, 2004; 556(2): 623 - 635. [Abstract] [Full Text] [PDF]