HUMAN, MOUSE, AND RAT CALNEXIN CDNA CLONING - IDENTIFICATION OF POTENTIAL CALCIUM-BINDING MOTIFS AND GENE LOCALIZATION TO HUMAN-CHROMOSOME-5

Calnexin is a 90-kDa integral membrane protein of the endoplasmic reti culum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. We have purified human calnexin in association with the human interferon -gamma receptor and cloned calnexin cDNA from placenta. Fragments of c alnexin have been prepared as glutathione S-transferase fusion protein s and analyzed for their abilities to bind Ca-45(2+) and ruthenium red . A subdomain containing four internal repeats binds Ca2+ with the hig hest affinity. This sequence is highly conserved when compared to calr eticulin (a luminal ER protein), an Onchocerca surface antigen, and ye ast and plant calnexin homologues. Consequently, this sequence represe nts a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the ''E-F hand'' motif. An adjacent subdomain, a lso highly conserved and containing four internal repeats, fails to bi nd Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic intera ctions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. We have also determined the cDNA sequences of mouse and rat calnexins. Comparison of the known mammalian calnexi n sequences reveals very high conservation of sequence identity (93-98 %), suggesting that calnexin performs important cellular functions. Th e gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35.