Elimination of Differences in the Mobility of Flax Isoperoxidases on PAGE by Digestion with α-Mannosidase

Abstract

ABSTRACIIn the flax (Linum usitatissimum) genotype Stormont cirrus, anodic peroxidases from the genotroph S migrate more slowly on PAGE and SDS-PAGE than the corresponding peroxidases from the genotroph L. When purified isoperoxidases S2 and L2 were digested with a-mannosidase, the difference in mobility was eliminated. Treatment with a-fucosidase and j8-xylosidase also altered the mobility of S2 and L2, but affected the sensitivity to the action of endo-O-N-acetylglucosaminidase H of only S2. Our results suggest differences in posttranslational processing of the carbohydrate moiety between S and L isoperoxidases. These differences were also found in other S and L glycoenzymes (anodic acid phosphatases) as well as in the peroxidases of other flax genotypes.Durrant (8,9) Rm value of the L type (11, 13). These observations suggest that the Rm shifts are the result of posttranslational modifications since it is unlikely that changes across all possible structural gene loci for these enzymes would yield unidirectional Rm shifts. The absence ofco-dominance in F, hybrids ofL and S for all isozymes of peroxidase (24) also points to differences in posttranslational processing. An obvious common site for posttranslational modification of both the peroxidase and acid phosphatase isozymes in genotrophs L and S is their carbohydrate moiety since they all are glycoproteins (14). This is further supported by the recent observations that corresponding L and S isoperoxidases can be chromatographically separated from one another on columns of Sepharose-bound Con A (15) but not by isoelectric focusing nor can they be distinguished from one another by immunochemical methods (16).In this report, we present results from investigations into the structure of the carbohydrate moiety of purified L and S peroxidase isozymes using column chromatography on several Sepharose-bound lectins with different sugar affinities and the effect of various glycosidase digestions on the Rm difference between L and S peroxidase and acid phosphatase isozymes. The anodic peroxidase pattern in several other flax genotypes was also examined along with the effect of a-mannosidase treatment on their migration on PAGE. Our results suggest that the Rm differences between L and S glycoenzymes are due to higher levels of mannosylation of these proteins in the genotroph S. MATERIALS AND METHODS

Cloning and Molecular Characterization of the Basic Peroxidase Isoenzyme from Zinnia elegans, an Enzyme Involved in Lignin Biosynthesis

“…4) with an estimated M r (MALDI-TOF) of 31,460. That is, the heterogeneity of the Z. elegans peroxidases may be attributed to the heterogeneity of the glycan moieties, as has previously been reported for peroxidases from flax (Gaudreault and Tyson, 1988), barley (Rasmussen et al, 1991;Johansson et al, 1992), and peanut (Wan et al, 1994). This assumption was further strengthened by the total similarity of the N-terminal sequence determined for both peroxidases (LSTTFYDTT), and by the 99.9% similarity of the tryptic fragment fingerprints obtained by RP nano-LC and MALDI-TOF MS (Fig.…”

Charge and molecular weight differences for fourteen guaiacol peroxidase isozymes in flax indicate that most are encoded by different structural genes

“…Some heterogeneity resulting from differences in weight had been anticipated because peroxidase isozymes with heterogeneous carbohydrate moieties have been reported in flax [19] as well as in other species such as petunia [20] and peanut [21]. The isozymes of the A.Prx 4 complex have always seemed likely candidates for this type of heterogeneity.…”

Heritable changes in electrophoretic properties of flax peroxidases resulting from variation in N nutrient level

“…L isozymes apparently all undergo additional elimination of mannosyl residues from their oligosaccharide chains compared to the corresponding isozymes in S. Evidence from affinity chromatography and the in vitro action of various glycosidases indicates that additional mannosyl residues are processed out of the L forms of isozymes 1, 2 and 4, whereas in S these mannosyl residues are left untouched. This suggests differences in the activity/synthesis of a main stem amannosidase between L and S. These studies are described in detail elsewhere Gaudreault & Tyson, 1988). …”

“…This suggests underlying differences in post-translational (or transcriptional ) processing of the oligosaccharide chains of the L and S glycoproteins. Using purified peroxidase isozymes, immunochemical comparisons, affinity to Con A columns and the effects of glycosidases on Rm Gaudreault & Tyson, 1988) also point to differences in carbohydrate components in the corresponding L and S peroxidases. In vivo, S peroxidases apparently retain mannose residues that are processed out of the corresponding L forms; amannosidase will in vitro remove these mannose residues from purified L and S peroxidases so that the Rm shift between L and S isozymes is eliminated .…”