Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187.
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 approximately 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.
Kalra
Seema
S
Department of Medicine, Medical College of Wisconsin and Veterans Adminstration Medical Center, Milwaukee, WI 53295, USA.
Li
Ning
N
Seetharam
Shakuntla
S
Alpers
David H
DH
Seetharam
Bellur
B
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003037:Cobalt Radioisotopes; D017124:Conserved Sequence; D003470:Culture Media / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D066298:In Vitro Techniques; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D016296:Mutagenesis; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D012867:Skin / Q000166:cytology; D013329:Structure-Activity Relationship; D013462:Sulfur Radioisotopes; D014155:Transcobalamins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014805:Vitamin B 12 / Q000378:metabolism*
D003037:Cobalt Radioisotopes; D003470:Culture Media; D004220:Disulfides; D009097:Multienzyme Complexes; D011956:Receptors, Cell Surface; D013462:Sulfur Radioisotopes; D014155:Transcobalamins; C028176:transcobalamin receptor; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D003545:Cysteine; D014805:Vitamin B 12