The stability of yeast invertase is not significantly influenced by glycosylation.

N Schülke,F X Schmid

doi:10.1016/s0021-9258(18)68381-4

Abstract

Yeast invertase exists in two different forms. The cytoplasmic enzyme is nonglycosylated, whereas the external invertase contains about 50% carbohydrate of the high mannose type. The protein moieties of both enzymes are identical. The two invertases have been used previously as a model system to investigate the influence of covalently linked carbohydrate chains on the stability of large glycoproteins, and controversial results were obtained. Here, we measured thermal and denaturant-induced unfolding by various probes, such as the loss of enzymatic activity, and by the changes in absorbance and fluorescence. The ranges of stability of the two invertases were found to be essentially identical, indicating that the presence of a high amount of carbohydrate does not significantly contribute to the stability of external invertase. Earlier findings that invertase is stabilized by glycosylation could not be confirmed. The stability of this glycoprotein is apparently determined by the specific interactions of the folded polypeptide chain. Unlike the glycosylated form, the carbohydrate-free invertase is prone to aggregation in the denatured state at high temperature and in a partially unfolded form in the presence of intermediate concentrations of guanidinium chloride.

Highlights

Yeast invertase exists in two different forms
Most of the proteins which are transported along the secretory pathway become glycosylated in thecourse of synthesis and/or transport (Sharon and Lis, 1982)
N-Glycosylation is a cotranslational event; prefabricated oligosaccharide unitsaretransferred from the lipid carrier dolichyl diphosphate to Asn residues as soon as the growing polypeptide chain enters the lumen of the endoplasmic reticulum (Rothman andLodish, 1975; Kiely et al, 1976)

Summary

Introduction

Yeast invertase exists in two different forms. The Ashwell,1980; but see Schwaiger et al, 1982), for the cytoplasmicenzyme is nonglycosylated, whereas the biological function (Kalyan and Bahl, 1983; Breitfeld et al, external invertase contains about 50% carbohydrate 1984;Berman et al, 1985;Olden et al, 1982),and for receptorof the high mannose type. N-Glycosylation is a cotranslational event; prefabricated oligosaccharide unitsaretransferred from the lipid carrier dolichyl diphosphate to Asn residues as soon as the growing polypeptide chain enters the lumen of the endoplasmic reticulum (Rothman andLodish, 1975; Kiely et al, 1976) This suggests that N-glycosylation precedes the folding andmaturation of the nascent glycoprotein toits native state. The carbohydrate content of external invertase is high; it amounts to about 50%.The cytoplasmic form of invertase is free of carbohydrate Both proteins are the product of the same (SUC2) gene, i.e. they have identical protein moieties with a subunit M , of about 58,000 (Perlman and Halvorson, 1981; Taussig and Carlson, 1983). The only sequence difference lies in the presence of an additional Nterminal serineresidue present inthe external protein (Taussig and Carlson, 1983).Because of the availability of both the glycosylated and carbohydrate-free forms, chemical or enzymatic deglycosylation procedures arenot required.

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Results

Conclusion