Isolation and sequence analysis of variant forms of human transcobalamin II.
Two cDNA clones (1.9 kb and 1.5 kb, respectively) encoding full length human TC II have been isolated from a human endothelial cell cDNA library and sequenced. The differences between the two clones are the length of the 5' end and the 3' end non-coding regions and the codon at position 198 and 219. Both the clones differ from the recently isolated (human endothelial cell) cDNA for TC II (Platica, O., Janecko, R., Quadros, E.V., Regee, A., Romain, R. and Rothenberg, S.P. (1991) J. Biol. Chem. 266, 7860-7863) in codon 259 and 376 and in their calculated pI values. In vitro transcription followed by translation in a reticulocyte lysate system and SDS-PAGE revealed that the isolated cDNA clones encode a protein of 43 kDa. Upon treatment with canine pancreatic microsomes, the molecular mass of the in vitro translated product was reduced to 41.5 kDa, indicating the presence of an approximately 1.5 kDa signal peptide. This translation product was immunoprecipitated with rabbit anti-serum to human TC II and was able to bind to Cbl-Sepharose beads. The amino acid sequence alignment of TC II with that of other Cbl binding proteins (rat intrinsic factor, human transcobalamin I and porcine haptocorrin) revealed only 33% overall homology. However, there were four regions of greater than 80% homology and two regions of about 60% homology. These regions encompass the majority of the hydrophobic areas of the Cbl-binders. Based on these studies, we suggest that structural basis for the expression of different polymorphic forms of TC II may be due to single point mutations and that TC II, like other mammalian Cbl-binders, have evolved from a common ancestral gene. Furthermore, the Cbl-binding functional domain most probably resides in a hydrophobic pocket which is formed by all or some of the six regions of high homology.
Li
N
N
Division of Gastroenterology, Medical College of Wisconsin, Milwaukee 53226.
Seetharam
S
S
Lindemans
J
J
Alpers
D H
DH
Arwert
F
F
Seetharam
B
B
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics; D004730:Endothelium, Vascular / Q000502:physiology; D015723:Gene Library; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014155:Transcobalamins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014471:Umbilical Veins
D003062:Codon; D014155:Transcobalamins; D004247:DNA