Synthesis of a human lysosomal enzyme, beta-hexosaminidase B, using the baculovirus expression system.
Human lysosomal beta-hexosaminidase exists in two major forms: the A isoform is composed of both alpha and beta chains, while the B form is a homopolymer of beta chains. Deficiency of beta-hexosaminidase underlies the GM2 gangliosidoses. We have produced active beta-hexosaminidase B in cultured insect (Sf9) cells by isolation of a recombinant insect virus (baculovirus) containing the cDNA for the beta chain within the viral polyhedron gene and infection of Sf9 cells with this construct. That portion of the enzyme secreted into the medium, 50%, was purified with concanavalin A Sepharose and subsequent affinity chromatography to yield beta-hexosaminidase B that is 75% pure. The product has an N-terminal amino acid sequence, specific activity, and size (M(r) 62,000) similar to that of the enzyme present in cultured human fibroblasts. However, endo H sensitivity studies revealed that the oligosaccharide structures present on recombinant beta-hexosaminidase B differ from those found on the enzyme synthesized in the human system. In addition, these structures lack the mannose 6-phosphate recognition marker that targets degradative hydrolases to lysosomes. Despite these differences, recombinant beta-hexosaminidase B does serve as a specific substrate for the mannose phosphorylating enzyme, N-acetylglucosaminyl phosphotransferase. Furthermore, the oligosaccharide moieties phosphorylated in vitro match those phosphorylated in vivo, pointing to the conformational integrity of the recombinant enzyme. Generous amounts of easily obtained, easily purified, and properly folded beta-hexosaminidase B will facilitate physical structural analysis of the enzyme.
Boose
J A
JA
Laboratory of Biochemistry and Metabolism, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Tifft
C J
CJ
Proia
R L
RL
Myerowitz
R
R
D016367:Baculoviridae / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D006801:Humans; D008247:Lysosomes / Q000201:enzymology; D008360:Mannosephosphates / Q000737:chemistry; D008970:Molecular Weight; D010770:Phosphotransferases / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups); D014357:Trypsin; D001619:beta-N-Acetylhexosaminidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D008360:Mannosephosphates; D011994:Recombinant Proteins; C027693:mannose-6-phosphate; D004247:DNA; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D001619:beta-N-Acetylhexosaminidases; D014357:Trypsin