Cloning and expression of gamma-glutamyl transpeptidase from isolated porcine brain capillaries.
In order to understand the mechanisms regulating the blood-brain barrier we isolated a cDNA clone for gamma-glutamyl transpeptidase from purified porcine brain capillaries by cross-species DNA hybridization using a DNA fragment from a rat kidney cDNA clone. The sequence of the porcine gamma-glutamyl transpeptidase cDNA shows 84% identity in the coding region to the sequence of human placenta gamma-glutamyl transpeptidase cDNA. The derived protein sequences of the human and porcine gamma-glutamyl transpeptidase are 98% similar. Alignment of the porcine and the published rat gamma-glutamyl transpeptidase cDNA sequences revealed an average identity of 79%, except for a small region of 194 base pairs containing a high number of mismatches. After four nucleotide changes within this region of the published rat cDNA sequence, which correct two frame shifts, the derived protein sequences of the rat and the porcine gamma-glutamyl transpeptidase show 97% similarity. The corrected sequence, which includes a region of 65 amino acids, contains additional N-glycosylation sites as well as a hydrophobic protein domain that might represent a second membrane-spanning domain. Tissue-specific expression and diversity of the gamma-glutamyl transpeptidase mRNA transcripts in various porcine and human tissues were analysed by Northern blot hybridization. The 2.2-kb transcript of brain gamma-glutamyl transpeptidase is mainly expressed in the endothelial cells of brain microvessels, in agreement with previous data that showed the brain protein being localized within the blood-brain barrier.
Papandrikopoulou
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A
Institut für Biochemie, Technische Hochschule Darmstadt, Federal Republic of Germany.
Frey
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A
Gassen
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002196:Capillaries / Q000201:enzymology; D002560:Cerebrovascular Circulation; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005260:Female; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D005723:gamma-Glutamyltransferase / Q000235:genetics*
D012313:RNA; D004247:DNA; D005723:gamma-Glutamyltransferase