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Glycobiology Advance Access published online on January 26, 2005
Glycobiology, doi:10.1093/glycob/cwi045
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© Oxford University Press 2004; all rights reserved.
Received September 20, 2004
Revised January 10, 2005
Accepted January 19, 2005

Article

Intact {alpha}-1,2-Endomannasidase is a typical type-II membrane protein

Stephen R. Hamilton 1, Huijuan Li 1, Harry Wischnewski 1, Anita Prasad 2, Joanna S. Kerley-Hamilton 3, Teresa Mitchell 1, Amelia J. Walling 2, Robert C. Davidson 1, Stefan Wildt 1, and Tillman U. Gerngross 2*

1 GlycoFi Inc., 21 Lafayette St, Lebanon, NH 30766, USA
2 Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA
3 Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755, USA

* To whom correspondence should be addressed.
Tillman U. Gerngross, E-mail: tillman.gerngross{at}dartmouth.edu


  Abstract

Rat endomannosidase is a glycosidic enzyme that catalyzes the cleavage of di-, tri-, or tetrasaccharides (Glc1-3Man), 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 homologues, 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 the current report we have cloned two novel endomannosidase sequences from rat and human cDNA libraries, but were unable to amplify the ORF of the previously reported rat sequence. Analysis of the rat genome confirmed that the 5’- and 3’-termini of the previously reported sequence were in fact located on different chromosomes. This in combination with out inability to amplify the previously reported sequence indicated that the rat endomannosidase sequence previously published was likely a cloning artifact, and that the sequences reported in the current study encode the intact protein. 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.

Keywords: glycosylation/ glycosidase/ Pichia pastoris/ recombinant expression/ transmembrance domain.
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