Cloning, expression, purification, and characterization of the murine lysosomal acid alpha-mannosidase.
Catabolism of alpha-linked mannose residues on eukaryotic glycoproteins is accomplished by a broad specificity lysosomal alpha-mannosidase (EC 3.2.1.24). Based on regions of protein sequence conservation between the lysosomal alpha-mannosidase from Dictyostelium discoideum and the murine Golgi glycoprotein processing alpha 1,3/1,6-mannosidase, alpha-mannosidase II, we have cloned a cDNA encoding the murine lysosomal alpha-mannosidase. The longest of the clones was 3.1 kb in length and encoded a polypeptide of 992 amino acids containing a putative NH2-terminal signal sequence and 11 potential N-glycosylation sites. The deduced amino acid sequence was 76.5% identical to the human lysosomal alpha-mannosidase and 38.1% identical to the lysosomal alpha-mannosidase from D. discoideum. Expression of the cDNA in Pichia pastoris resulted in the secretion of an alpha-mannosidase activity into the culture medium. This recombinant expression product was purified and was shown to have enzymatic characteristics highly similar to the enzyme purified from mammalian sources and to the human lysosomal alpha-mannosidase cDNA expressed in Pichia. These characteristics include a similar pH optimum, Km, Vmax, inhibition by swainsonine, and activity toward natural substrates. Northern blots identified a major 3.5 kb RNA transcript in all murine tissues tested. A minor transcript of 5.4 kb was also detected in some murine tissues similar to the alternatively spliced transcripts that have been previously identified in human tissues.
Merkle
R K
RK
Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
Zhang
Y
Y
Ruest
P J
PJ
Lal
A
A
Liao
Y F
YF
Moremen
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D006023:Glycoproteins / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D043323:alpha-Mannosidase
D018076:DNA, Complementary; D006023:Glycoproteins; D011994:Recombinant Proteins; D008361:Mannosidases; D043323:alpha-Mannosidase