Methods and compositions for detecting and modulating O-glycosylation

• US 8,993,718 B2
• Filed: 08/29/2013
• Issued: 03/31/2015
• Est. Priority Date: 06/15/2007
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Gross, Benjamin J., Kahne, Suzanne Walker, Swoboda, Jonathan G.
• Primary Examiner(s): Shen, Bin
• Application Number:
  US14/013,860
• Publication Number:
  US 20140187444A1
• Time in Patent Office: 579 Days
• Field of Search: 530/300
• US Class Current: 530/300
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12N 9/12 transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)
• CPC Class Codes: G01N 2440/38 addition of carbohydrates, e.g. glycosylation, glycation
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Abstract

The invention relates to methods and products for modulating glycosylation of proteins. The invention is useful for identifying therapeutic compounds to treat glycosylation-associated disorders such as neurodegeneration, diabetes, including complications of diabetes such as insulin resistance, nephropathy, microvascular damage, and endothelial dysfunction. The invention is also useful for identifying therapeutic compounds to treat de-glycosylation-associated disorders such as ischemic damage and traumatic injury. The invention also relates in part to assays that are useful for identifying and testing candidate compounds for modulating glycosylation of proteins and also relates in part to compounds to treat glycosylation-associated diseases and disorders.

Claims

1. An isolated molecule comprising a cleavable detectably labeled polypeptide substrate having

a) an O-glycosylation site and

b) a specific protease cleavage site positioned in the polypeptide such that the rate of cleavage by a specific protease at the specific protease cleavage site is different when the polypeptide is glycosylated at the O-glycosylation site than when the polypeptide is not glycosylated at the O-glycosylation site, wherein the detectable label is positioned in the polypeptide such that a change in the detectable label identifies cleavage of the polypeptide by the specific protease at the specific protease cleavage site.

2. The isolated molecule of claim 1, wherein the polypeptide O-glycosylation site is a serine or a threonine residue.

3. The isolated molecule of claim 2, wherein the serine or threonine is positioned within 1, 2, 3, or 4 amino acids of the specific protease cleavage site.

4. The isolated molecule of claim 1, wherein the specific protease cleavage site is a single site and there is only one specific protease cleavage site.

5. The isolated molecule of claim 1, wherein the specific protease cleavage site is positioned on the N-terminal side of the O-glycosylation site.

6. The isolated molecule of claim 1, wherein the specific protease cleavage site is positioned on the C-terminal side of the O-glycosylation site.

7. The isolated molecule of claim 1, wherein the detectable label comprises a fluorescent, enzyme, radioactive, metallic, biotin, chemiluminescent, or bioluminescent molecule.

8. The isolated molecule of claim 1, wherein the detectable label is a fluorescent moiety.

9. The isolated molecule of claim 8, wherein the change in the detectable label comprises a change in fluorescence of the fluorescent moiety.

10. The isolated molecule of claim 1, wherein the detectable label is a FRET donor and a FRET acceptor pair.

11. The isolated molecule of claim 10, wherein the FRET donor and acceptor pair is 7-diethylaminocoumarin-3-carboxylic acid (DEAC) and fluorescein isothiocyanate;

[2-(1-sulfonyl-5-naphthyl)-aminoethylamide] EDANS and 4,4-dimethylazobenzene-4′

carbonyl (DABCYL);

fluorescein isothiocyanate and tetramethylrhodamine (TMR);

or Tryptophan or tyrosine and dinitrophenyl moieties.

12. The isolated molecule of claim 8, wherein the fluorescent moiety is a Cy5.5 emitter or FITC, Texas red, tetramethylrhodamine (TMR), AlexaFluor dyes, HiLyte Fluorophores, or [2-(1-sulfonyl-5-naphthyl)-aminoethylamide] EDANS.

13. The isolated molecule of claim 1, wherein the polypeptide further comprises a quenching moiety.

14. The isolated molecule of claim 13, wherein the quenching moiety is QXL-520, BHQ3, Iowa black, 4,4-dimethylazobenzene-4′

carbonyl (DABCYL), BHQ1, BHQ10, QXL-570, QXL-620, dinitrophenyl (DNP) containing groups, or QSY-7,9-21, or 35.

15. The isolated molecule of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:

STPVSSANMK (SEQ ID NO;

1), STPVSFANMK (SEQ ID NO;

2), STPVSRANMK (SEQ ID NO;

3), EYIPTVFDNK (SEQ ID NO;

4), EYRPTVFDNK (SEQ ID NO;

5), EYIPTVDDNK (SEQ ID NO;

6), EPGPTEAPK (SEQ ID NO;

25), and EDAVTPGPK (SEQ ID NO;

26).

16. The isolated molecule of claim 1, wherein the specific protease cleavage site is a proteinase K cleavage site, a trypsin cleavage site, a chymotrypsin cleavage site, a thermolysin cleavage site, Staphylococcal peptidase I cleavage site, Proline-endopeptidase cleavage site, Pepsin cleavage site, Glutamyl endopeptidase cleavage site, Factor Xa cleavage site, Granzyme B Lysyl endopeptidase cleavage site, Asp-N Endopeptidase cleavage site, or enterokinase cleavage site.

17. A kit for identifying an agent that modulates O-glycosylation, the kit comprising a package housing a first container containing a polypeptide of claim 1, and instructions for using the polypeptide to identify modulators of O-glycosylation.

18. The kit of claim 17, further comprising a second container containing the specific protease that cleaves at the specific protease site of the polypeptide.

19. The kit of claim 17, wherein the polypeptide comprises the amino acid sequence set forth as one of SEQ ID NOs:

1-6, 25, or 26.