Endomannosidases in the modification of glycoproteins in eukaryotes

Abstract

The present invention generally relates to methods of modifying the glycosylation structures of recombinant proteins expressed in fungi or other lower eukaryotes, to more closely resemble the glycosylation of proteins from higher mammals, in particular humans. The present invention also relates to novel enzymes and, nucleic acids encoding them and, hosts engineered to express the enzymes, methods for producing modified glycoproteins in hosts and modified glycoproteins so produced.

1 - 13 . (canceled) 
     
     
         14 . A host cell comprising a nucleic acid encoding a chimeric endomannosidase comprising the catalytic domain of an endomannosidase fused to a cellular targeting peptide to target the endomannosidase to the ER or Golgi of the host cell. 
     
     
         15 . The host cell of  claim 14  wherein the host cell is a mammalian, plant, insect, fungal, yeast, algal or bacterial cell. 
     
     
         16 . The host cell of  claim 14 , wherein the host cell is selected from the group consisting of  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia  sp.,  Saccharomyces cerevisiae, Saccharomyces  sp.,  Hansenula polymorpha, Kluyveromyces  sp.,  Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium  sp.,  Fusarium gramineum, Fusarium venenatum  and  Neurospora crassa.    
     
     
         17 . A method for modifying glycosylation structures in a lower eukaryote comprising: expressing an endomannosidase activity in the lower eukaryote a chimeric endomannosidase comprising the catalytic domain of an endomannosidase fused to a cellular targeting peptide to target the endomannosidase to the ER or Golgi of the lower eukaryote wherein the endomannosidase activity removes a composition comprising at least a glucose residue and one mannose residue on an oligosaccharide. 
     
     
         18 . The method of  claim 17  wherein the endomannosidase activity further comprises the activity of truncating Glc 1-3 Man 9-5 GlcNAc 2  to Man 8-4 GlcNAc 2  wherein Glcα1,3Man, Glc 2 α1,3Man or Glc 3 α1,3Man are removed. 
     
     
         19 . The method of  claim 17  wherein the endomannosidase activity comprises hydrolysis of a composition comprising at least one glucose residue and one mannose residue on glucosylated glycans. 
     
     
         20 . The method of  claim 17  wherein the endomannosidase introduced are targeted to the endoplasmic reticulum, the early, medial, late Golgi, trans Golgi network or any vesicular compartment within the host organism. 
     
     
         21 . The method of  claim 17  wherein the endomannosidase is of host origin but has been modified by mutation, promoter strength or copy number to enhance activity. 
     
     
         22 . The method of  claim 17  wherein the endomannosidase is secreted. 
     
     
         23 . The method of  claim 17  wherein the host cell is a mammalian, plant, insect, fungal, yeast, algal or bacterial cell. 
     
     
         24 . The method of  claim 17  wherein the lower eukaryote is selected from the group consisting of  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia  sp.,  Saccharomyces cerevisiae, Saccharomyces  sp.,  Hansenula polymorpha, Kluyveromyces  sp.,  Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium  sp.,  Fusarium gramineum, Fusarium venenatum  and  Neurospora crassa.    
     
     
         25 . A method for modifying glucosylated glycoproteins comprising introducing an endomannosidase activity into a lower eukaryotic host cell wherein upon expression of the endomannosidase activity modifies a glucosylated glycoprotein that has bypassed the ER.