Methods and compositions for detections and modulating O-glycosylation
First Claim
1. A method of detecting the level of O-glycosylation of a polypeptide, the method comprising:
- (a) contacting a glucosyltransferase enzyme and a sugar donor with a molecule comprising a detectably labeled polypeptide having;
(i) an O-glycosylation site and(ii) a specific protease cleavage site positioned in the polypeptide such that the rate of cleavage of the polypeptide by the specific protease at the specific protease cleavage site is different when the polypeptide is O-glycosylated at the glycosylation site than when the polypeptide is not O-glycosylated at the glycosylation site,wherein the detectable label is positioned in the polypeptide such that a change in the amount of detectable label correlates with cleavage of the polypeptide by the specific protease;
(b) adding to the contacted polypeptide the specific protease that cleaves at the specific protease cleavage site of the polypeptide; and
,(c) monitoring the rate of cleavage of the polypeptide, thereby detecting the level of O-glycosylation of the polypeptide.
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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.
60 Citations
7 Claims
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1. A method of detecting the level of O-glycosylation of a polypeptide, the method comprising:
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(a) contacting a glucosyltransferase enzyme and a sugar donor with a molecule comprising a detectably labeled polypeptide having; (i) an O-glycosylation site and (ii) a specific protease cleavage site positioned in the polypeptide such that the rate of cleavage of the polypeptide by the specific protease at the specific protease cleavage site is different when the polypeptide is O-glycosylated at the glycosylation site than when the polypeptide is not O-glycosylated at the glycosylation site, wherein the detectable label is positioned in the polypeptide such that a change in the amount of detectable label correlates with cleavage of the polypeptide by the specific protease; (b) adding to the contacted polypeptide the specific protease that cleaves at the specific protease cleavage site of the polypeptide; and
,(c) monitoring the rate of cleavage of the polypeptide, thereby detecting the level of O-glycosylation of the polypeptide. - View Dependent Claims (2, 3, 4)
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5. A method of identifying a compound that modulates polypeptide O-glycosylation, the method comprising:
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(a) contacting a glucosyltransferase enzyme, a sugar donor, and a candidate modulating compound with a molecule comprising a detectably labeled polypeptide having; (i) an O-glycosylation site and (ii) a specific protease cleavage site positioned in the polypeptide such that the rate of specific protease cleavage of the polypeptide glycosylated at the O-glycosylation site is different than the rate of specific protease cleavage of the polypeptide not glycosylated at the O-glycosylation site, wherein the detectable label is positioned in the polypeptide such that a change in the amount of detectable label correlates with cleavage of the polypeptide by the specific protease at the specific protease cleavage site; (b) adding to the contacted polypeptide the specific protease; (c) monitoring the rate of specific protease cleavage of the polypeptide; and
,(d) comparing the rate of specific protease cleavage of the polypeptide to a control rate of specific protease cleavage wherein a difference in the control rate of specific protease cleavage compared to the rate of cleavage of the polypeptide contacted with the candidate modulating compound identifies the candidate modulating compound as modulating O-glycosylation of the polypeptide. - View Dependent Claims (6, 7)
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Specification