Glycoprotein and process for producing the same
A process for producing a lysosomal enzyme having a mannose-6-phosphate-containing acidic sugar chain, wherein the process comprising: culturing in a medium yeast cells obtained by introducing a lysosomal enzyme gene into a sugar chain biosynthetic enzyme gene mutant strain of yeast, collecting a lysosomal enzyme having a phosphate-containing sugar chain from the culture, and then treating the enzyme with α-mannosidase; and pharmaceutical compositions for treatment of human lysosomal enzyme deficiencies produced by the process. The genetic engineering technique using the yeast according to the present invention allows large-amount and high-purity production of a glycoprotein having a phosphate-containing acidic sugar chain which can serve as a labeling marker for transporting into lysosomes in cells of mammals such as human. The glycoprotein having a phosphate-containing acidic sugar chain according to the invention may be utilized as a drug effective in treatment of human lysosomal enzyme deficiencies, etc.
1. A process for producing an active form of a glycoprotein comprising an acidic sugar chain having a mannose-6-phosphate at a non-reducing end, comprising
(i) expressing a gene introduced into yeast encoding said glycoprotein; and
(ii) treating said glycoprotein with α-mannosidase obtained from Cellulomonas SO-5 strain bacteria (FERM BP-7628) to remove a mannose residue from the mannose- 1-phosphate in the sugar chain of the glycoprotein,
wherein a-1,6-mannosyltransferase gene and a-1,3-mannosyltransferase gene have been disrupted in said yeast.
2. The process of claim 1 , wherein said acidic sugar chain binds to mannose-6-phosphate receptor.
3. The process of claim 1 , wherein said acidic sugar chain is a high mannose-type sugar chain consisting of the structure of one of structural formulas I to VII:
4. The process of claim 1 , wherein the α-1,6-mannosyltransferase gene is the OCH1 gene of S. cerevisiae and the α-1,3-mannosyltransferase gene is the MNN1 gene of S. cerevisiae.
5. The process of claim 1 , wherein the mutant yeast has a highly phosphorylated sugar chain.
6. The process of claim 1 , wherein the active form of a glycoprotein having an acidic sugar chain having a mannose-6-phosphate is a lysosomal enzyme.
7. The process of claim 6 , wherein the lysosomal enzyme is α-galactosidase.
8. The process of claim 7 , wherein the α-galactosidase is human α-galactosidase.
9. The process of claim 8 , wherein the gene encoding the α-galactosidase comprises the nucleotide sequence of SEQ ID. No: 5.
10. The process of claim 1 , wherein the c˜-mannosidase has an activity which non-specifically degrades α-1,2-mannoside linkages, α-1,3-mannoside linkages and α-1,6-mannoside linkages.
11. The process of claim 1 , wherein the c˜-mannosidase has an exo activity, and comprises no endo activity.