Molecular cloning of chicken calcyclin (S100A6) and identification of putative isoforms.
A full-length cDNA encoding smooth muscle calcyclin (S100A6) was cloned from chicken gizzard, using reverse transcription--polymerase chain reaction techniques. The deduced amino acid sequence contains 92 residues with 12 substitutions and a 2 amino acid C-terminal extension when compared with human calcyclin. Calcyclin was purified from chicken gizzard by Ca(2+)-dependent hydrophobic chromatography, heat treatment, and anion-exchange chromatography, N-terminal sequencing of two CNBr peptides confirmed its identity as calcyclin. Two isoforms of calcyclin (A and B), which differ with respect to the presence or absence of a C-terminal lysine, were identified and the native protein was shown to exist as noncovalently associated homodimers (AA and BB) and heterodimers (AB). Incubation of purified calcyclin AA with an extract of chicken gizzard did not result in degradation of calcyclin A or appearance of calcyclin B, suggesting that calcyclin B is a bona fide isoform rather than a proteolytic fragment generated during purification. Western blotting of chicken tissues with anti-(gizzard calcyclin) indicated abundant expression of calcyclin in smooth muscle tissues, including esophagus, large intestine, and trachea, with lower levels in lung, heart, kidney, and brain, and none detectable in liver or skeletal muscle.
Allen
B G
BG
Department of Medical Biochemistry, University of Calgary, AB, Canada.
Andrea
J E
JE
Sutherland
C
C
Schönekess
B O
BO
Walsh
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D018797:Cell Cycle Proteins; D002645:Chickens / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D005895:Gizzard, Avian / Q000737:chemistry; D007536:Isomerism; D051379:Mice; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000737:chemistry; D009928:Organ Specificity; D011817:Rabbits; D051381:Rats; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; C489133:S100A6 protein, Gallus gallus; C489134:S100a6 protein, mouse; C489132:S100a6 protein, rat