Abstract
Collectrin, a novel homolog of angiotensin-converting enzyme-related carboxypeptidase (ACE2), was identified during polymerase chain reaction-based cDNA subtraction and up-regulated in 5/6 ablated kidneys at hypertrophic phase. Collectrin, with 222 amino acids, has an apparent signal peptide and a transmembrane domain; the sequence is conserved in mouse, rat, and human and shares 81.9% identity. Human collectrin has 47.8% identity with non-catalytic extracellular, transmembrane, and cytosolic domains of ACE2; however, unlike ACE and ACE2, collectrin lacks active dipeptidyl carboxypeptidase catalytic domains. The collectrin mRNA transcripts are expressed exclusively in the kidney. In situ hybridization reveals its mRNA expression in renal collecting ducts, and immunohistochemistry shows that it is localized to the luminal surface and cytoplasm of collecting ducts. Immunoprecipitation studies, using [35S]methionine-labeled renal cortical and inner medullar collecting duct cells, i.e. M-1 and mIMCD-3, indicate that the protein size is approximately 32 kDa. During the development of mouse kidney, mRNA signal is detectable at day 13 of gestation, and the protein product is observed in the ureteric bud branches. Its expression is progressively increased during later stages of the gestation extending into the neonatal periods and then is decreased in adult life. Up-regulated expression of collectrin in the hypertrophic kidneys after renal ablation and restricted spatio-temporal expression during development indicates a possible role(s)in the process of progressive renal failure and renal organogenesis.
Highlights
Collectrin, a novel homolog of angiotensin-converting enzyme-related carboxypeptidase (ACE2), was identified during polymerase chain reaction-based cDNA subtraction and up-regulated in 5/6 ablated kidneys at hypertrophic phase
1181 bp of rat collectrin (AF178086) and 1345 bp of human collectrin (AF229179) cDNAs were isolated by rapid amplification of 5Ј- and 3Ј-cDNA ends (RACE)-PCR (Fig. 1B)
A ClustalW multiple alignment indicates that the primary protein structure of mouse, rat, and human collectrin was highly conserved throughout the entire sequence (Fig. 1B)
Summary
Isolation of a Full-length Collectrin cDNA and Nucleotide Sequencing—Using cDNA-RDA, we previously described the isolation of differentially expressed genes in the 5/6 nephrectomized mouse remnant kidney [3, 4]. Among the various novel genes, NX-17 (collectrin partiallength cDNA) had a ϳ2-fold up-regulated mRNA expression in nephrectomized mouse with 5/6 ablated kidney [5]. Using this partial-length cDNA, the full coding sequence of collectrin cDNA from mouse, rat, and human was isolated with the Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA). KGPPGV, the sequence of which was derived from the amino acid stretch between residues 165 and 179 of the intracellular domain of collectrin (Fig. 1A). This segment of collectrin is highly conserved and identical between the mouse and rat.
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