The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure

Abstract

The heat-and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75°C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety. In contrast, the mature form, which lacks the propeptide composed of 91 amino acid residues, showed more complex behaviour: differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition. These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarboxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding. The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding.Keywords: carboxypeptidase Y; fluorescence spectrometry; pressure unfolding; procarboxypeptidase Y; thermal unfolding.Carboxypeptidase Y (CPY), a member of the serine carboxypeptidase family, is a 61-kDa vacuolar enzyme obtained from Saccharomyces cerevisiae [1]. This enzyme is synthesized in the form of procarboxypeptidase Y (pro-CPY) and sorted to the vacuole via the Golgi apparatus where it undergoes carbohydrate modification. ProCPY has an N-terminal extension (propeptide) of 91 residues [2,3], compared to the mature CPY. This propeptide structure is essential for folding both in vivo and in vitro, as well as for maintaining CPY in an inactive form [4][5][6]. The mature and precursor forms are glycoproteins [7], which contain % 16% carbohydrates [8]: the four carbohydrate chains are of similar sizes and are bound to asparagine residues at Asn-Xaa-Thr glycosylation sites [9][10][11][12]. The genetic replacement of these asparagine residues by alanine residues produces unglycosylated (Dgly) CPY [13] and proCPY with no change in their activities.The reason why the presence of the propeptide is important for the correct folding of CPY and role that the large amount of carbohydrate moiety plays on the stability and function of CPY has not been fully clarified. To answer these questions, we examined the folding/unfolding of mature and precursor CPY as well as their unglycosylated forms using temperature and pressure as the structural perturbant. Compared to heat, pressure studies have been used to obtain complementary information concerning protein-solvent interactions [14,15], the unfolded states of proteins [16,17], and protein folding pathways [18].Our analytical techniques involved the use of differential scanning calorimetry (DSC) and protein fluorescence as a function of temperature and pressure. The intrinsic fluorescence of CPY is due mainly to tryptophan and, to a lesser extent, tyrosine residues [19]. The shape and the wavelength of the emission...