Molecular cloning of porcine soluble angiotensin-binding protein.

N Sugiura,H Hagiwara,S Hirose

doi:10.1016/s0021-9258(19)37153-4

Abstract

Soluble angiotensin-binding protein (sABP) is a 75-kDa cytosolic protein that binds angiotensins and its analogues with high affinity. In this study, the primary structure of porcine sABP is determined by cDNA cloning. Based on the partial amino acid sequences of sABP tryptic fragments, fully degenerate oligonucleotides were synthesized, and used as primers for polymerase chain reactions to amplify the corresponding sABP cDNA fragment from porcine liver first-strand cDNA. By using initially the polymerase chain reaction product and later partial cDNA clones as probes, porcine heart and liver cDNA libraries were screened, and several positive clones were obtained including one covering the entire coding region. From the cDNA sequence, an open reading frame that encodes sABP as a 704-amino acid protein with molecular mass of 80,800 daltons is predicted. No significant homology was seen between sABP and other proteins in GenBank and NBRF data bases, including the angiotensin-related proteins such as angiotensin converting enzyme, renin, and AT1 angiotensin II receptor. Northern blot analysis of poly(A)+ RNA revealed that the mRNA for sABP is expressed as 5.3- and 2.8-3.2-kilobase transcripts. These transcripts are generated by the use of alternative polyadenylation signals. Within the 3'-untranslated region of the cDNA sequence downstream from the polyadenylation signals for smaller transcripts, a porcine short interspersed repetitive element (SINE) was found; only the longer 5.3-kilobase transcript had the SINE sequence.

Highlights

Soluble angiotensin-binding protein is a 75- the majorcardiovascular andhydromineral effects hasrekDa cytosolic protein that binds angiotensins and its cently beencloned from vascular smooth muscle cells [19]
In this study cDNA clones for porcine Soluble angiotensin-binding protein (sABP) were isolated, and the aminoacid sequence of sABP was deduced from their nucleotide sequences. sABP is a cytosolic protein that binds angiotensins andits peptide analogues with high affinity comparable tothat of the membrane-boundangiotensin receptors [23, 25]
The deduced amino acid sequence of sABP, sharesno homology with the angiotensin receptor which has recently been cloned by expression cloning, shown to be a member of the heptahelical receptorfamily, and classified as the subtype ATI[19, 20] based on its selectivity for non-peptide angiotensin antagonist losartan (DuP 753; Ref. 48)

Summary

RESULTS

Amino Acid Sequences of Tryptic Fragments of sABP-. The probe used for screening was the PCR-amplified 450-bp sABP Since initial attempts todetermine the N-terminal sequence cDNA fragment (Fig. 2) labeled with [a-"'PIdCTP (Amersham) by the random primer method [31]. Duplicate plaque lifts were prehybridized and hybridized at 60 "C in 6 X SSPE (1 X SSPE is 0.15 M NaCl, mM NaH,PO, (pH 7.0), 1 mM EDTA), X Denhardt's solution Amino acid sequences of tryptic fragments of sABP portion of the XPAB4 (nucleotides 1349-1365) was used as primer in Tryptic fragments of sABP separated by reverse phase HPLC were the first-strand synthesis. 205-217 contained an unrelated sequence 3' to the sABP cDNA, probably representing double insert recombinant

STHHVTAFLDDLSQK VDQSLHTNTSLDAASEYAK

AA T

IPABl A

DISCUSSION