Hydrolysis of mannose-1-phospho-6-mannose linkage to phospho-6-mannose

A method of producing a target protein having an uncapped phospho-6-mannose residue, said method comprising:

• providing a fungal cell genetically engineered to comprise and express a nucleic acid encoding a mannosidase, said mannosidase being capable of hydrolyzing a mannose-1-phospho-6-mannose residue to phospho-6-mannose;
• and
• introducing into said cell a nucleic acid encoding a target protein, wherein said cell produces said target protein in which a mannose-1-phospho-6-mannose residue on the target has been converted to a phospho-6-mannose residue by said expressed mannosidase expressed in the cell.

A method of producing a target protein having an uncapped phospho-6-mannose residue in a fungal organism, said method comprising

• a) providing a fungal cell genetically engineered to comprise and express a nucleic acid encoding a mannosidase capable of hydrolyzing mannose-1-phospho-6-mannose to phospho-6-mannose, and said fungal cell further comprising a nucleic acid encoding a target protein in which a mannose-1-phospho-6-mannose residue on the target has been converted to a phospho-6-mannose residue by said expressed mannosidase expressed in the cell;
• and
• b) isolating said target protein having said uncapped phospho-6-mannose residue.

An isolated fungal cell genetically engineered to produce glycoproteins comprising an uncapped phospho-6-mannose residue, said fungal cell comprising a nucleic acid encoding a mannosidase, wherein expression of said mannosidase in said fungal cell results in the production of glycoproteins comprising said terminal phospho-6-mannose residues, wherein said mannosidase is a member of glycosyl hydrolase family 92.

A substantially pure culture of Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Arxula adeninivorans, Pichia methanolica, Oogataea minuta, or Aspergillus niger cells, a substantial number of which are genetically engineered to produce a glycoprotein comprising an uncapped phospho-6-mannose residue, said cells comprising a nucleic acid encoding a mannosidase capable of hydrolyzing mannose-1-phospho-6-mannose to phospho-6-mannose, wherein said mannosidase is a member of glycosyl hydrolase family 92.

A composition comprising a glycoprotein, wherein at least 47% of the N-glycans on said glycoprotein comprise an uncapped phospho-6-mannose residue.

A polypeptide comprising an amino acid sequence having at least 90% identity to residues 1 to 774 of SEQ ID NO:50 or to SEQ ID NO:50, wherein the polypeptide is capable of hydrolyzing mannose-1-phospho-6-mannose to phospho-6-mannose.