The Effect of Cysteine Modifications Kinetics of Paraoxonase-1 and Glycosylation of PON1

The Effect of Cysteine Modifications Kinetics of Paraoxonase-1 and Glycosylation of PON1

Bibliographic Details
Main Author: Lokko, Kaarina
Language:English
Published: The Ohio State University / OhioLINK 2020
Subjects:
Chemistry
Chemical Engineering
Biochemistry
PON1 glycosylation paraoxonase1 glutathionylation
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=osu1578070312150032
id ndltd-OhioLink-oai-etd.ohiolink.edu-osu1578070312150032
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Chemistry
Chemical Engineering
Biochemistry
PON1 glycosylation paraoxonase1 glutathionylation
spellingShingle Chemistry
Chemical Engineering
Biochemistry
PON1 glycosylation paraoxonase1 glutathionylation
Lokko, Kaarina
The Effect of Cysteine Modifications Kinetics of Paraoxonase-1 and Glycosylation of PON1
author Lokko, Kaarina
author_facet Lokko, Kaarina
author_sort Lokko, Kaarina
title The Effect of Cysteine Modifications Kinetics of Paraoxonase-1 and Glycosylation of PON1
title_short The Effect of Cysteine Modifications Kinetics of Paraoxonase-1 and Glycosylation of PON1
title_full The Effect of Cysteine Modifications Kinetics of Paraoxonase-1 and Glycosylation of PON1
title_fullStr The Effect of Cysteine Modifications Kinetics of Paraoxonase-1 and Glycosylation of PON1
title_full_unstemmed The Effect of Cysteine Modifications Kinetics of Paraoxonase-1 and Glycosylation of PON1
title_sort effect of cysteine modifications kinetics of paraoxonase-1 and glycosylation of pon1
publisher The Ohio State University / OhioLINK
publishDate 2020
url http://rave.ohiolink.edu/etdc/view?acc_num=osu1578070312150032
work_keys_str_mv AT lokkokaarina theeffectofcysteinemodificationskineticsofparaoxonase1andglycosylationofpon1
AT lokkokaarina effectofcysteinemodificationskineticsofparaoxonase1andglycosylationofpon1
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-osu15780703121500322021-08-03T07:13:46Z The Effect of Cysteine Modifications Kinetics of Paraoxonase-1 and Glycosylation of PON1 Lokko, Kaarina Chemistry Chemical Engineering Biochemistry PON1 glycosylation paraoxonase1 glutathionylation One potential route to treatment for intoxication by chemical agents is to catalytically destroy the toxic species. Hydrolytic enzymes from several species have been suggested for this purpose, but mammalian proteins are especially attractive because they are less likely to elicit an immunological response. Secreted proteins from eukaryotes are often glycosylated and often contain disulfide bonds for stability, but these features also present challenges for development of therapeutics. Chemical modification by polyethylene glycol (PEG) or other moieties can be used to simulate natural modification or alter pharmacokinetic properties of proteins in serum.Paraoxonase-1 (PON1) is a serum mammalian protein that hydrolyzes a wide array of substrates including lactone, esters, and organophosphorus (OP) compounds. Its weak OPase activity has made it a possible lead for enzymic countermeasures against OP toxins. PON1 has three conserved cysteine residues, two of which are generally thought to form a disulfide, and two sites of N-glycosylation. We sought to generate a mutant of PON1 for selective PEGylation, to control its pharmacokinetic properties in serum. We found that even low levels of non-specific PEGylation by NHS ester led to inactivation, so we proceeded to examine the effects of Cys modification. Using several different engineered backgrounds—G2E6, G3C9, 4E9, and 1-I-F11—we examined the effects of Cys mutation, reduction, glutathione incubation, and PEGylation on the activity and stability of the protein. Surprisingly, we were able to engineer Cys-free variants with wild-type like activity and only moderate reductions in thermal stability. Also, in contrast to previous reports on human PON1 and G3C9, we found that incubation with large amounts of reduced or oxidized glutathione resulted in little change in activity. We present evidence that PON1 may not have a disulfide bond formed in the folded state. We generated variants with Asn to Cys mutations at putative sites of N-glycosylation in mammalian cells, resulting in selective PEGylation with a maleimide in one case, but we found that the mutants lacked catalytic activity. These results raise interesting questions about the role of Cys residues in PON1’s biological function and highlight challenges in engineering sites for selective modification of therapeutically interesting proteins.Glycosylation is an important biological function carried out by many organisms. The function of a protein or enzyme can be affected by the glycosylation pattern it exhibits, and disease states are often caused by abnormal glycosylation. Glycosylation is important for the production, transport, stabilization, protection against proteases and overall function of many proteins. In this study, the aim was to determine which sites of PON1 are naturally glycosylated and their glycosylation states/patterns to glean a better picture of the function of glycosylation in PON1. To this end, several systems were designed for the expression of PON1 in mammalian cells.Chemical glycosylation allows for the increased lifetime of a protein. PON1was chemically glycosylated and analyzed for activity. The glycans used for the chemical glycosylation was unique in that they were harvested from naturally occurring sources. High-mannose polysaccharides from soy protein and RNAse B glycans were used to demonstrate the labeling of polysaccharide for HPLC analysis and detection by MALDI. High-mannose glycan was also successfully labeled for conjugation to PON1. Likewise, PEGylated PON1 was purified and analyzed before sending for animal studies. First the protein was expressed and then chemically reacted with glycans specifically designed to react with the cysteines in the protein, then tested for sustained reactivity. PON1 glycosylated in a Tris buffer system maintained activity comparable to PON1 that was not glycosylated. The chemically glycosylated PON1 was sent for animal testing to be compared to PEGylated PON1. 2020-10-12 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1578070312150032 http://rave.ohiolink.edu/etdc/view?acc_num=osu1578070312150032 restricted--full text unavailable until 2025-05-13 This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

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