Enzymes and methods for cleaving N-glycans from glycoproteins

Patent metadata

Field	Value
Publication number	US-9327016-B2
Application number	US-201314378190-A
Country	US
Kind code	B2
Filing date	Feb 14, 2013
Priority date	Feb 14, 2012
Publication date	May 3, 2016
Grant date	May 3, 2016

Abstract

Provided herein are deglycosylating enzymes that remove a broad range of N-glycans from N-glycosylated proteins. Further provided are methods of recombinantly producing and expressing the deglycosylating enzymes. The presently described deglycosylating enzymes can be used to produce free glycans for characterization, and for prebiotic and immunostimulatory uses. In addition, the presently described deglycosylating enzymes can be used to produce deglycosylated proteins for characterization, to improve digestion, and to reduce immunogenicity.

First claim

What is claimed is:

1. A method of increasing the efficiency of protein digestion in an individual, comprising administering a pharmaceutical composition comprising a recombinant polypeptide comprising a sequence at least 90% identical to SEQ ID NO:5, wherein the polypeptide can cleave high mannose, complex, and hybrid N-glycans from a glycoprotein, and wherein the polypeptide lacks a transmembrane domain to the individual, thereby increasing the efficiency of protein digestion in the individual.

2. The method of claim 1 , wherein the individual is an infant.

3. A method of inducing satiety in an individual, comprising administering a pharmaceutical composition comprising a recombinant polypeptide comprising a sequence at least 90% identical to SEQ ID NO:5, wherein the polypeptide can cleave high mannose, complex, and hybrid N-glycans from a glycoprotein, and wherein the polypeptide lacks a transmembrane domain to the individual, thereby inducing satiety in the individual.

4. A method of deglycosylating a glycoprotein comprising a high mannose, complex, or hybrid N-glycan, the method comprising
contacting the glycoprotein with a polypeptide comprising a sequence at least 90% identical to SEQ ID NO:5, wherein the polypeptide lacks a transmembrane domain, thereby deglycosylating the glycoprotein and generating deglycosylated protein and free glycans.

5. A method of recombinantly producing a polypeptide, wherein said polypeptide comprises a sequence at least 90% identical to SEQ ID NO:5,
wherein said polypeptide can cleave high mannose, complex, and hybrid N-glycans from a glycoprotein,
the method comprising culturing a cell comprising a recombinant polynucleotide encoding the polypeptide comprising a sequence at least 90% identical to SEQ ID NO:5 under conditions appropriate for expression of the polypeptide, and
contacting the polypeptide with a glycoprotein comprising a high mannose, complex, or hybrid N-glycan, thereby deglycosylating the glycoprotein, thereby recombinantly producing the polypeptide.

6. The method of claim 5 , wherein the polypeptide lacks a transmembrane domain that spans a cell membrane.

7. The method of claim 5 , further comprising isolating the polypeptide.

8. The method of claim 5 , wherein the glycoprotein is selected from the group consisting of: lactoferrin, whey, and immunoglobulin.

9. A composition comprising:
(i) a recombinant polypeptide comprising a sequence at least 90% identical to or SEQ ID NO:5, wherein said polypeptide can cleave high mannose, complex, and hybrid N-glycans from a glycoprotein and wherein the polypeptide lacks a transmembrane domain; and
(ii) a glycoprotein, wherein the glycoprotein comprises a high mannose, complex, or hybrid N-glycan.

10. The composition of claim 9 , wherein the glycoprotein is selected from the group consisting of: lactoferrin, whey, and immunoglobulin.

11. The composition of claim 9 , wherein the N-glycan comprises core fucosylation, terminal fucosylation, or terminal sialylation.

12. The composition of claim 9 , wherein the polypeptide is a transmembrane protein in a cell membrane protein in a cell.

13. The composition of claim 9 , wherein the glycoprotein comprises a high mannose N-glycan.

14. The method of claim 4 , wherein the glycoprotein comprises a high mannose N-glycan.

15. The method of claim 4 , wherein the glycoprotein comprises a complex N-glycan.

16. The method of claim 4 , wherein the glycoprotein comprises a hybrid N-glycan.

Assignees

• Univ California

Inventors

• Garrido Daniel
• German J Bruce
• Lebrilla Carlito B
• Mills David A

Classifications

• A61P37/00

  Drugs for immunological or allergic disorders · CPC title

• A23L33/195

  Proteins from microorganisms · CPC title

• A23C9/1216

  Other enzymes · CPC title

• C12P21/005

  Glycopeptides, glycoproteins · CPC title

• A61K38/40

  Transferrins, e.g. lactoferrins, ovotransferrins · CPC title