Rabbit small intestinal trehalase. Purification, cDNA cloning, expression, and verification of glycosylphosphatidylinositol anchoring.
alpha,alpha-Trehalase (EC 3.2.1.28), an intrinsic protein of intestinal brush-border membranes, was purified to homogeneity from rabbits. Partial amino acid sequences were determined. Two degenerate oligonucleotides based on the sequence of a CNBr peptide were employed in a polymerase chain reaction to amplify a 71-base pair fragment of trehalase DNA with rabbit intestine cDNA as a starting template. This fragment was used as a hybridization probe to isolate full length trehalase clones from a rabbit intestine cDNA bank. Sequence analysis revealed that trehalase comprises 578 amino acids, contains at the amino terminus a typical cleavable signal sequence, at the carboxyl terminus a rather hydrophobic region typical of proteins anchored via glycosylphosphatidylinositol, and four potential N-glycosylation sites. Trehalase has no sequence homologies with other sequenced brush-border glycosidases. Northern blot analysis revealed a 1.9-kilobase trehalase mRNA in small intestine and kidney, smaller amounts in liver, and none in lung. Southern blot analysis indicated the gene has a length of 20 kilobase pairs or less. Injection into Xenopus laevis oocytes of mRNA synthesized in vitro from a trehalase template resulted in the expression of trehalase activity several hundredfold above background. The trehalase activity was membrane-bound and could be solubilized upon digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. This strongly suggests that rabbit small intestinal trehalase is anchored via glycosylphosphatidylinositol also when expressed in X. laevis oocytes.
Ruf
J
J
Department of Biochemistry II, Swiss Federal Institute of Technology, Eidgenössische Technische Hochschule-Zentrum, Zürich, Switzerland.
Wacker
H
H
James
P
P
Maffia
M
M
Seiler
P
P
Galand
G
G
von Kieckebusch
A
A
Semenza
G
G
Matei
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D017886:Durapatite; D015870:Gene Expression; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D006882:Hydroxyapatites; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology; D010716:Phosphatidylinositols / Q000378:metabolism*; D011061:Poly A / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D011817:Rabbits; D014158:Transcription, Genetic; D014198:Trehalase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D006882:Hydroxyapatites; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D017886:Durapatite; D014198:Trehalase