Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family.

TLDR

CHIP28 is a 28‑kDa integral membrane protein found in erythrocytes and renal tubules that shares homology with the ancient family of water‑permeable channel proteins. The CHIP28 cDNA was isolated by a three‑step PCR strategy from human fetal liver, then used to recover a full‑length clone from a bone marrow library, and its sequence was verified by expression and immunoblotting. The full‑length cDNA encodes a 3.1‑kb transcript with an 807‑bp ORF that predicts six transmembrane domains, two N‑glycosylation sites, and intracellular termini, and its sequence shows strong homology to the bovine lens major intrinsic protein, suggesting CHIP28 functions as a member of the ancient water‑channel family.

Abstract

CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5' untranslated nucleotide sequence, an 807-bp open reading frame, and approximately 2 kilobases of 3' untranslated sequence containing a polyadenylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DNA sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.