Molecular cloning of a cDNA encoding human calumenin, expression in Escherichia coli and analysis of its Ca2+-binding activity.
By microsequencing and cDNA cloning we have identified the transformation-sensitive protein No. IEF SSP 9302 as the human homologue of calumenin. The nucleotide sequence predicts a 315 amino acid protein with high identity to murine and rat calumenin. The deduced protein contains a 19 amino acid N-terminal signal sequence, 7 EF-hand domains and, at the C-terminus, a HDEF sequence which has been reported to function as retrieval signal to the ER. The calumenin transcript is ubiquitously expressed in human tissue, at high levels in heart, placenta and skeletal muscle, at lower levels in lung, kidney and pancreas and at very low levels in brain and liver. Calumenin belongs to a family of multiple EF-hand proteins that include the ER localized proteins reticulocalbin and ERC-55 and the Golgi localized Cab45. Since its Ca2+ binding may be important for the function of the protein we have used microdialysis experiments in order to analyse for the affinity and the capacity of recombinant human (rh) calumenin. All 7 EF-hands of the protein are functional and bind Ca2+, each with an affinity of 1.6x103 M-1. The relatively low affinity for the EF-hands may suggest a role for the protein in Ca2+-dependent processes in the ER.
Vorum
H
H
Department of Medical Biochemistry, Ole Worms Allé, Building 170, University of Aarhus, DK-8000 Aarhus C, Denmark.
Liu
X
X
Madsen
P
P
Rasmussen
H H
HH
Honoré
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D000090204:Mesothelin; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
C510527:CALU protein, human; D002135:Calcium-Binding Proteins; C107182:Calu protein, mouse; D018076:DNA, Complementary; C000715529:Msln protein, rat; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000090204:Mesothelin; D002118:Calcium