3. PURIFICATION AND BIOPHYSICAL CHARACTERIZATION OF SECRETED
3.5 GENERAL DISCUSSION
3.5.2 Apase purification and physiochemical characterization
Results from ion-exchange chromatography revealed two acid phosphatase peaks (Figure 3.4).
Unfortunately, the apase were isolated in relatively low yields. In order to address this problem, protease inhibitors were added to protein samples with the intent to inhibit the suspected enzymatic degradation by proteases. The yields did not improve. The low protein yield is reported to be a problem often encountered with lichen proteins (PRINTZEN et al., 2003).
Thus, the nature of the second protein remains to be characterized. When two techniques were used in succession, enzyme activity was reduced dramatically. After several attempts, gel filtration was the best choice for purification. The prevalent apase from Cladonia portentosa was purified 45-fold to near electrophoretic homogenetic and final pNPP-hydrolyzing specific unity (167 units/mg) (Table 3.5). The purified enzyme was estimated by non-denaturing PAGE to be approximately 250 kDa (Figure 3.7A). When a product was denatured, a molecular size of 148 kDa was obtained (Figure 3.8 A).
Adding the reducing agents to the purified enzyme, and analysis by SDS-PAGE followed by activity staining, revealed two protein staining bands with molecular masses of approximately 116,25 and 36 kDa, respectively (Figure 3.8B). The activity of the enzyme was not destroyed by the addition of the reducing agents. These data suggest that the native enzyme could exist as a heterodimer. Dissociation of the enzyme into subunits was obtained only after treatment with reducing agents (either 2-mercaptoethano or DTT) indicating that disulfide bridges are included in association of the subunits of this enzyme (HOGG, 2003). Five disulfide bonds were predicted to exist in Aspergillus spp. (ULLAH and MULLANEY, 1996). Using the site-
122 directed mutagenesis RODRIGUEZ et al. (2000) demonstrated that disulfide bonds were important for catalytic activity and conformation of acid phosphatase enzymes.
Considerable heterogeneity with regards to apase subunit structure, varying from monomeric to hetero-dimer and tetramer forms have been reported on several acid phosphatases from filamentous fungi. In Penicillium funiculosun the phosphatase had a 76 kDa heterodimer composed of 51 and 26 kDa subunits, when quantified on SDS-PAGE (YOSHIDA et al., 1989a). It was found that acid phosphatase from Schizosaccharomyces pombe exists as a dimer- tetramer dissociating mono-equilibriating system with a dimer of 180 kDa (DIBENEDETTO and TELLER, 1981). In constrast, apase in Candida albicans was reported to be a monomer with Mr 131 kDa (ODDS and HIERHOLZER, 1973). Aspergillus niger pH 2.5 apase has an apparent native molecular mass of 269 kDa and it also forms a tetramer in solution (KOSTREWA et al., 1999).
Taken together, the eletrophoretic patterns of purified enzyme (native-PAGE and SDS-PAGE), fit the profile of high molecular weight acid phosphatase and thus this enzyme can be assigned to this class (ARNOLD et al., 1988; VINCENT et al., 1992). The estimated molecular weight by SDS-PAGE (148 kDa) was, however, higher than that observed in other filamentous fungi of the Ascomycete family. The diffusing nature of this enzyme was attributed to glycosylation (WANNET et al., 2000; MULLANEY et al., 2000). It has been estimated that about a third of all proteins that enter secretory pathways in eukaryotic cells may be N-glycosylated and so tens of thousands of glycoprotein variants may coexist in eukaryotic cells (WALSH et al., 2005).
Thus, the discrepancy between the two estimations may be due to an overestimation of molecular mass by SDS-PAGE which is common in the context of glycoprotein (DECEDUE et al., 1984). The variation in the extent of glycosylation of a single gene product may be associated with the regulation or targeting proteins to fulfill distinct roles (WEBER and PITT, 1997b).
In addition, the sequencing data show that the apparent strong homologies to existing proteins relate to translated products of much lower expected MW‟s, suggesting that the protein/s
123 purified in this study were perhaps significantly modified (e.g. glycosylation). Again leading to an indistinct band pattern, or that they have become complexed in such a way that normal SDS- PAGE did not dissociate them to their natural apparent MW‟s.
PAS staining did not reveal a clear staining of glycoprotein, although the band was stained margenta after periodic stain. The colour was lost after subsequent washing steps. The removal of bound carbohydrates by endo-F collapsed the major apase into proteins of lower molecular weight (Figure 3.13). Electrophoretic pattern by SDS-PAGE analysis of N-glycosidas F-treated enzyme revealed ~78 kDa reduction in apparent molecular mass after deglycosylation.
Reduction of apase in size after deglycosylation to approximated 70 kDa under denatured conditions was consistent with glycosylation reported in secreted protein in most filamentous fungi. In Aspergillus caespitosus, the purified enzymes were glycoproteins showing 63.0 and 58.3 % of carbohydrate content respectively (GUIMARÃES et al., 2004). The molecular weight of the glycosylated pH 2.5 acid phosphatase produced by a A. niger ALKO243 is about 66 kDa and the endo-F treated deglycosylation is around 47 kDa (MIETTINEN-OINONEN et al., 1997). Aspergillus niger pH 2.5 apase has an apparent native molecular mass of 269 kDa with a glycosylated subunit of approximately 65 kDa and an unglycosylated form of 50.8 kDa (KOSTREWA et al., 1999).
Interestingly, the purified enzyme from C. portentosa was stable at room temperature even after several days and was not degraded by proteases, probably due to the high amount of attached sugar. The stability of the purified enzyme has long been attributed to the glycosylic nature of the protein, a phenomenon observed in several glycosylated proteins (ULLAH, 1993). A high degree of glycosylation is presumed to protect enzymes from attack by proteases (BERKA et al., 1991; KUBICEK et al., 1993; KLIONSKY et al., 1990). ULLAH (1993) suggested that glyco-conjugates present in acid phosphatase may even prevent the protease from degrading the peptide bonds. The results further revealed that deglycosylation resulted in a significant loss of acid phosphate activity, susceptibility to proteolytic degradation and denaturing at higher temperatures (ULLAH, 1993). Similar results were noted by HAN and LEI, (1999) in apase and fungal xylanase (VAN DE VYVER et al., 2004). When xylanase was exposed to proteases or rumen fluid in vitro, the enzyme demonstrated stability, it was obvious that glycosylation
124 enhances stability, thus, contributing towards the stability of exogenous enzymes (VAN DE VYVER et al., 2004).
The pI of purified protein in Cladonia portentosa was estimated to be 6.4 under the given experimental conditions (Figure 3.9). Most fungal acid phosphatase displayed acid pI such as that of A. niger NRRL3135 which was reported to be 4.0 (HA et al., 1999) and in A. ficuum, the unglycosylated protein had a pI of 4.97 (ULLAH, 1998). The phytases produced by A. niger (NRRL 3135) PhyA and PhyB were secretory glycoprotein with pI 4.5 and 4.9 respectively (ULLAH, 1998). In Bacillus subtillis a pI of 6.5 was obtained (KEROVUO et al., 1998).