Introduction: Alpha-2-macroglobulin (α2m) is a plasma glycoprotein serine protease inhibitor. Previous studies have shown that coagulation factor concentrations are highly variable with age and α2m levels have been found to be twice as high in newborns compared to adults. This may contribute to a resistance towards thrombotic events observed in young populations. Protein glycosylation is known to affect protein activity and the glycosylation profile of adult α2m has previously been analyzed. Information regarding glycosylation of α2m in other age groups has yet to be elucidated. Therefore, the purpose of this study is to examine the differences in the glycosylation profiles between newborn and adult α2m.
Methods: To evaluate glycan macroheterogeneity, plasma samples were enzymatically deglycosylated by PNGaseF, followed by SDS PAGE and western blotting (WB) to detect α2m. To evaluate microheterogeneity, plasma samples were incubated with Neuraminidase (Clostridium perfringens) followed by native PAGE and WB to determine sialic acid content. To detect non-sialylated terminal galactose residues, plasma samples were incubated with immobilized RCA120, and lectin-bound molecules were separated from unbound molecules. Additionally, the affinity of α2m for ricin was evaluated by eluting bound proteins with increasing concentrations of galactose. All fractions were subjected to SDS-PAGE and WB to detect α2m. 2D gel electrophoresis was completed to examine differences in pI and molecular weight of α2m in both age groups. Purification by immunoprecipitation was also performed and eluted α2m was analyzed by fluorescence-assisted carbohydrate electrophoresis (FACE) to determine the glycan fingerprint in the two populations.
Results: Deglycosylation of both newborn and adult α2m with PNGaseF resulted in a change in migration and apparent molecular weight, however no statistically significant difference was found between newborn and adult. On native PAGE following treatment with neuraminidase, newborn α2m exhibited a statistically significant change in migration compared to adult. Additionally, newborn α2m exhibited a higher percentage of molecules bound to RCA120 than adult (no statistical difference) and elution of α2m from RCA120 with a galactose step gradient produced similar profiles for newborn and adult molecules. 2D electrophoresis and WB revealed a difference in pI of α2m in newborns as compared to adults. Finally, purified newborn and adult α2m were analyzed by FACE and quantification of prominent fluorescent bands revealed a higher secondary:primary band ratio in newborns when compared to adults.
Conclusions: To our knowledge, this is the first study investigating glycosylation differences between newborn and adult α2m molecules. The results from PNGaseF analyses indicate no significant difference in total N-glycan content. Neuraminidase results suggest significantly greater sialic acid presence on newborn α2m, however there was no significant difference in galactose content. 2D electrophoresis revealed a difference in pI as well as the way in which newborn and adult α2m degrade when exposed to experimental conditions. A2m was successfully purified from both newborn and adult plasma, and FACE results indicate that the proportion of more branched glycans present in the two major fluorescent bands are of higher quantity in newborns than adults. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21077 |
| Date | January 2017 |
| Creators | Calvert, Laura |
| Contributors | Chan, Anthony, Medical Sciences (Blood and Cardiovascular) |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0018 seconds