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.
Department of Biochemistry II, Swiss Federal Institute of Technology, Eidgenössische Technische Hochschule-Zentrum, Zürich, Switzerland.
J
J
Ruf
H
H
Wacker
P
P
James
M
M
Maffia
P
P
Seiler
G
G
Galand
A
A
von Kieckebusch
G
G
Semenza
N
N
Matei
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