title: Intact {alpha}-1,2-endomannosidase is a typical type II membrane protein.
abstract: Rat endomannosidase is a glycosidic enzyme that catalyzes the cleavage of di-, tri-, or tetrasaccharides (Glc(1-3)Man), from N-glycosylation intermediates with terminal glucose residues. To date it is the only characterized member of this class of endomannosidic enzymes. Although this protein has been demonstrated to localize to the Golgi lumenal membrane, the mechanism by which this occurs has not yet been determined. Using the rat endomannosidase sequence, we identified three homologs, one each in the human, mouse, and rat genomes. Alignment of the four encoded protein sequences demonstrated that the newly identified sequences are highly conserved but differed significantly at the N-terminus from the previously reported protein. In this study we have cloned two novel endomannosidase sequences from rat and human cDNA libraries, but were unable to amplify the open reading frame of the previously reported rat sequence. Analysis of the rat genome confirmed that the 59- and 39-termini of the previously reported sequence were in fact located on different chromosomes. This, in combination with our inability to amplify the previously reported sequence, indicated that the N-terminus of the rat endomannosidase sequence previously published was likely in error (a cloning artifact), and that the sequences reported in the current study encode the intact proteins. Furthermore, unlike the previous sequence, the three ORFs identified in this study encode proteins containing a single N-terminal transmembrane domain. Here we demonstrate that this region is responsible for Golgi localization and in doing so confirm that endomannosidase is a type II membrane protein, like the majority of other secretory pathway glycosylation enzymes.
authors.affiliation: GlycoFi, Inc., 21 Lafayette Street, Lebanon, NH 03766, USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hamilton
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Harry
authors.initials: H
authors.lastname: Wischnewski
authors.forename: Anita
authors.initials: A
authors.lastname: Prasad
authors.forename: Joanna S
authors.initials: JS
authors.lastname: Kerley-Hamilton
authors.forename: Teresa
authors.initials: T
authors.lastname: Mitchell
authors.forename: Amelia J
authors.initials: AJ
authors.lastname: Walling
authors.forename: Robert C
authors.initials: RC
authors.lastname: Davidson
authors.forename: Stefan
authors.initials: S
authors.lastname: Wildt
authors.forename: Tillman U
authors.initials: TU
authors.lastname: Gerngross
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008361:Mannosidases / Q000096:biosynthesis / Q000145:classification / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000145:classification / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment
chemical_list: D008565:Membrane Proteins; C505100:MANEA protein, human; D008361:Mannosidases; C505101:alpha-1-2-endomannosidase, rat