Exploring the ultrastructural localization and biosynthesis of beta(1,4)-galactan in Pinus radiata compression wood.

Softwood species such as pines react to gravitropic stimuli by producing compression wood, which unlike normal wood contains significant amounts of beta(1,4)-galactan. Currently, little is known regarding the biosynthesis or physiological function of this polymer or the regulation of its deposition. The subcellular location of beta(1,4)-galactan in developing tracheids was investigated in Pinus radiata D. Don using anti-beta(1,4)-galactan antibodies to gain insight into its possible physiological role in compression wood. beta(1,4)-Galactan was prominent and evenly distributed throughout the S2 layer of developing tracheid cell walls in P. radiata compression wood. In contrast, beta(1,4)-galactan was not detected in normal wood. Greatly reduced antibody labeling was observed in fully lignified compression wood tracheids, implying that lignification results in masking of the epitope. To begin to understand the biosynthesis of galactan and its regulation, an assay was developed to monitor the enzyme that elongates the beta(1,4)-galactan backbone in pine. A beta(1,4)-galactosyltransferase (GalT) activity capable of extending 2-aminopyridine-labeled galacto-oligosaccharides was found to be associated with microsomes. Digestion of the enzymatic products using a beta(1,4)-specific endogalactanase confirmed the production of beta(1,4)-galactan by this enzyme. This GalT activity was substantially higher in compression wood relative to normal wood. Characterization of the identified pine GalT enzyme activity revealed pH and temperature optima of 7.0 and 20 degrees C, respectively. The beta(1,4)-galactan produced by the pine GalT had a higher degree of polymerization than most pectic galactans found in angiosperms. This observation is consistent with the high degree of polymerization of the naturally occurring beta(1,4)-galactan in pine.

Family 47 alpha-mannosidases in N-glycan processing.

Alpha-mannosidases in eukaryotic cells are involved in both glycan biosynthetic reactions and glycan catabolism. Two broad families of enzymes have been identified that cleave terminal mannose linkages from Asn-linked oligosaccharides (Moremen, 2000), including the Class 1 mannosidases (CAZy GH family 47 (Henrissat and Bairoch, 1996)) of the early secretory pathway involved in the processing of N-glycans and quality control and the Class 2 mannosidases (CAZy family GH38 [Henrissat and Bairoch, 1996]) involved in glycoprotein biosynthesis or catabolism. Within the Class 1 family of alpha-mannosidases, three subfamilies of enzymes have been identified (Moremen, 2000). The endoplasmic reticulum (ER) alpha1,2-mannosidase I (ERManI) subfamily acts to cleave a single residue from Asn-linked glycans in the ER. The Golgi alpha-mannosidase I (GolgiManI) subfamily has at least three members in mammalian systems (Herscovics et al., 1994; Lal et al., 1994; Tremblay and Herscovics, 2000) involved in glycan maturation in the Golgi complex to form the Man(5)GlcNAc(2) processing intermediate. The third subfamily of GH47 proteins comprises the ER degradation, enhancing alpha-mannosidase-like proteins (EDEM proteins) (Helenius and Aebi, 2004; Hirao et al., 2006; Mast et al., 2005). These proteins have been proposed to accelerate the degradation of misfolded proteins in the lumen of the ER by a lectin function that leads to retrotranslocation to the cytosol and proteasomal degradation. Recent studies have also indicated that ERManI acts as a timer for initiation of glycoprotein degradation via the ubiquitin-proteasome pathway (Hosokawa et al., 2003; Wu et al., 2003). This article discusses methods for analysis of the GH47 alpha-mannosidases, including expression, purification, activity assays, generation of point mutants, and binding studies by surface plasmon resonance.

Characterization of a human core-specific lysosomal {alpha}1,6-mannosidase involved in N-glycan catabolism.

In humans and rodents, the lysosomal catabolism of core Man(3)GlcNAc(2) N-glycan structures is catalyzed by the concerted action of several exoglycosidases, including a broad specificity lysosomal alpha-mannosidase (LysMan), core-specific alpha1,6-mannosidase, beta-mannosidase, and cleavage at the reducing terminus by a di-N-acetylchitobiase. We describe here the first cloning, expression, purification, and characterization of a novel human glycosylhydrolase family 38 alpha-mannosidase with catalytic characteristics similar to those established previously for the core-specific alpha1,6-mannosidase (acidic pH optimum, inhibition by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol, and stimulation by Co(2+) and Zn(2+)). Substrate specificity studies comparing the novel human alpha-mannosidase with human LysMan revealed that the former enzyme efficiently cleaved only the alpha1-6mannose residue from Man(3)GlcNAc but not Man(3)GlcNAc(2) or other larger high mannose oligosaccharides, indicating a requirement for chitobiase action before alpha1,6-mannosidase activity. In contrast, LysMan cleaved all of the alpha-linked mannose residues from high mannose oligosaccharides except the core alpha1-6mannose residue. alpha1,6-Mannosidase transcripts were ubiquitously expressed in human tissues, and expressed sequence tag searches identified homologous sequences in murine, porcine, and canine databases. No expressed sequence tags were identified for bovine alpha1,6-mannosidase, despite the identification of two sequence homologs in the bovine genome. The lack of conservation in 5'-flanking sequences for the bovine alpha1,6-mannosidase genes may lead to defective transcription similar to transcription defects in the bovine chitobiase gene. These results suggest that the chitobiase and alpha1,6-mannosidase function in tandem for mammalian lysosomal N-glycan catabolism.

Human EDEM2, a novel homolog of family 47 glycosidases, is involved in ER-associated degradation of glycoproteins.

In the endoplasmic reticulum (ER), misfolded proteins are retrotranslocated to the cytosol and degraded by the proteasome in a process known as ER-associated degradation (ERAD). Early in this pathway, a proposed lumenal ER lectin, EDEM, recognizes misfolded glycoproteins in the ER, disengages the nascent molecules from the folding pathway, and facilitates their targeting for disposal. In humans there are a total of three EDEM homologs. The amino acid sequences of these proteins are different from other lectins but are closely related to the Class I mannosidases (family 47 glycosidases). In this study, we characterize one of the EDEM homologs from Homo sapiens, which we have termed EDEM2 (C20orf31). Using recombinantly generated EDEM2, no alpha-1,2 mannosidase activity was observed. In HEK293 cells, recombinant EDEM2 is localized to the ER where it can associate with misfolded alpha1-antitrypsin. Overexpression of EDEM2 accelerates the degradation of misfolded alpha1-antitrypsin, indicating that the protein is involved in ERAD.