The molecular characterization of a novel GH38 α-mannosidase from the crenarchaeon Sulfolobus solfataricus revealed its ability in de-mannosylating glycoproteins.
α-Mannosidases, important enzymes in the N-glycan processing and degradation in Eukaryotes, are frequently found in the genome of Bacteria and Archaea in which their function is still largely unknown. The α-mannosidase from the hyperthermophilic Crenarchaeon Sulfolobus solfataricus has been identified and purified from cellular extracts and its gene has been cloned and expressed in Escherichia coli. The gene, belonging to retaining GH38 mannosidases of the carbohydrate active enzyme classification, is abundantly expressed in this Archaeon. The purified α-mannosidase activity depends on a single Zn(2+) ion per subunit is inhibited by swainsonine with an IC(50) of 0.2 mM. The molecular characterization of the native and recombinant enzyme, named Ssα-man, showed that it is highly specific for α-mannosides and α(1,2), α(1,3), and α(1,6)-D-mannobioses. In addition, the enzyme is able to demannosylate Man(3)GlcNAc(2) and Man(7)GlcNAc(2) oligosaccharides commonly found in N-glycosylated proteins. More interestingly, Ssα-man removes mannose residues from the glycosidic moiety of the bovine pancreatic ribonuclease B, suggesting that it could process mannosylated proteins also in vivo. This is the first evidence that archaeal glycosidases are involved in the direct modification of glycoproteins.
Cobucci-Ponzano
Beatrice
B
Institute of Protein Biochemistry - Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy.
Conte
Fiorella
F
Strazzulli
Andrea
A
Capasso
Clemente
C
Fiume
Immacolata
I
Pocsfalvi
Gabriella
G
Rossi
Mosè
M
Moracci
Marco
M
D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D017026:Swainsonine / Q000494:pharmacology; D043323:alpha-Mannosidase / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic; C504335:pancreatic ribonuclease B, bovine; D043323:alpha-Mannosidase; D008358:Mannose; D017026:Swainsonine