Cloning, characterization, and tissue distribution of porcine SPAI, a protein with a transglutaminase substrate domain and the WAP motif.
The primary and gene structures and tissue distribution of porcine SPAI-2, a protein that belongs to the WAP protein superfamily and has a sodium-potassium ATPase inhibitory activity, were determined by molecular cloning and Northern analysis. A full-length cDNA clone was isolated from a porcine duodenum cDNA library. The cDNA insert encoded a polypeptide of 187 amino acids, which is composed of three domains: a hydrophobic presequence of 21 amino acids, a prosegment of 105 amino acids ending with Asp126, and the mature SPAI-2 sequence of 61 amino acids beginning with Pro127. The prosegment contained 16 repeats of a hexapeptide that is highly homologous to the repetitive sequence found in the transglutaminase domain of the human elafin, an elastase-specific inhibitor that also belongs to the WAP superfamily. The repetitive sequence was demonstrated to be a good substrate of transglutaminase using a recombinant preparation produced in Escherichia coli. A porcine genomic library was then screened for the SPAI gene. Characterization and sequencing of positive clones indicated that the gene is similar to the elafin gene, having 3 exons encoding the 5'-untranslated region and signal sequence, proSPAI, and 3'-untranslated region, respectively. Northern blot analysis revealed intestine-specific expression of SPAI mRNA; the message was especially abundant in the small intestine. ProSPAI was also found in the circulation. The similarity of proSPAI to elafin in the domain structure, the acid-labile nature of the cleavage site (Asp126-Pro127), and the fact that the major form of SPAI in the plasma is proSPAI strongly suggest that proSPAI is not the precursor but rather it is the native form of SPAI. Like elafin, therefore, SPAI appears to be a new type of biologically active substance with a transglutaminase substrate domain that acts as an anchoring sequence.
Kuroki
J
J
Tsukuba Research Laboratories, Eisai Co., Ltd, Japan.
Hosoya
T
T
Itakura
M
M
Hirose
S
S
Tamechika
I
I
Yoshimoto
T
T
Ghoneim
M A
MA
Nara
K
K
Kato
A
A
Suzuki
Y
Y
Furukawa
M
M
Tachibana
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D036341:Intercellular Signaling Peptides and Proteins; D007413:Intestinal Mucosa / Q000378:metabolism; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013552:Swine; D014018:Tissue Distribution; D011503:Transglutaminases / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D036341:Intercellular Signaling Peptides and Proteins; D011506:Proteins; D012333:RNA, Messenger; C061402:SPAI-2 protein, Sus scrofa; D011503:Transglutaminases; D000254:Sodium-Potassium-Exchanging ATPase