Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa.
ABSTRACT Previous studies from our laboratory have demonstrated the presence of two integral proteins with glycosidase activity in the plasma membrane of Drosophila melanogaster spermatozoa and we have suggested that these enzymes might have a role in sperm-egg binding. In this study the glycosidases have been purified and characterized. We have evidenced the presence of three distinct enzymes, two beta-N-acetylhexosaminidase isoforms, named HEX 1 and HEX 2, and an alpha-mannosidase. The molecular size of the native enzymes estimated by gel filtration was 158 kDa for beta-hexosaminidases and 317 kDa for alpha-mannosidase. SDS-PAGE showed that HEX 1 and HEX 2 are dimers formed by subunits with different molecular sizes, whereas alpha-mannosidase consists of three subunits with different molecular weights. All the enzymes are terminally glycosylated. Characterization of the purified enzymes included their 4-methylumbelliferyl-substrate preferences, kinetic properties, inhibitor constants and thermal stability. On the basis of substrate specificity, kinetics and the results of inhibition studies, beta-hexosaminidases appear to differ from each other. HEX 1 and HEX 2 are similar to mammalian isoenzyme A and isoenzyme B, respectively. These findings represent the first report on the characterization of sperm proteins that are potentially involved in interactions with the egg in Insects.
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ABSTRACT: As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the functions of glycosyl hydrolases in the secretory pathway.PLoS Genetics 05/2014; 10(5):e1004349. DOI:10.1371/journal.pgen.1004349 · 8.17 Impact Factor
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ABSTRACT: The baculovirus-silkworm recombinant protein expression system is an excellent method for achieving high-level expression and post-translational modifications, especially glycosylation. However, the presence of paucimannosidic-type N-glycan in glycoproteins restricts their clinical use. Paucimannosidic-type N-glycan is produced by insect-specific membrane-binding-type β-N-acetylglucosaminidase (GlcNAcase). In the silkworm, BmGlcNAcase1, BmGlcNAcase2, and BmFDL are membrane-binding-type GlcNAcases. We investigated the localization of these GlcNAcases and found that BmFDL and BmGlcNAcase2 were mainly located in the fat body and hemolymph, respectively. The fat body is the main tissue of recombinant protein expression by baculovirus, and many glycoproteins are secreted into the hemolymph. These results suggest that inhibition of BmFDL and BmGlcNAcase2 could increase GlcNAc-type N-glycan levels. We therefore injected a GlcNAcase inhibitor into silkworms to investigate changes in the N-glycan structure of the glycoprotein expressed by baculovirus; modest levels of GlcNAc-type N-glycan were observed (0.8% of total N-glycan). Next, we generated a transgenic silkworm in which RNA interference (RNAi) reduced the BmFDL transcript level and enzyme activity to 25% and 50%, respectively, of that of the control silkworm. The proportion of GlcNAc-type N-glycan increased to 4.3% in the RNAi-transgenic silkworm. We conclude that the structure of N-glycan can be changed by inhibiting the GlcNAcases in silkworm.Journal of Bioscience and Bioengineering 09/2014; 119(2). DOI:10.1016/j.jbiosc.2014.07.012 · 1.79 Impact Factor
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ABSTRACT: β-N-acetylglucosaminidase (GlcNAcase) is a key enzyme in the chitin decomposition process. In this study, we investigated the gene expression profile of GlcNAcases and the regulation mechanism for one of these genes, BmGlcNAcase1, in the silkworm. We performed sequence analysis of GlcNAcase. Using dual-spike-in qPCR method, we examined the expression of Bombyx β-N-acetylglucosaminidases (BmGlcNAcases) in various tissues of silkworm as well as expression changes after stimulation with ecdysone. Using Bac-to-Bac system and luciferase reporter vectors, we further analyzed the promoter sequence of BmGlcNAcase1. The results showed that these proteins have a highly conserved catalytic domain. The expression levels of the BmGlcNAcase genes varied in different tissues, and were increased 48 h after exposure to ecdysone. BmGlcNAcase1 gene promoter with 5'-end serial deletions showed different levels of activity in various tissues, higher in the blood, skin and fat body. Deletion of the region from -347 to -223 upstream of BmGlcNAcase-1 gene abolished its promoter activity. This region contains the binding sites for key transcription factors including Hb, BR-C Z, the HSF and the typical TATA-box element. These results indicate that BmGlcNAcases are expressed at different levels in different tissues of the silkworm, but all are subjected to the regulation by ecdysone. BmGlcNAcase1 promoter analysis has paved a foundation for further study of the gene expression patterns.Molecular Biology Reports 07/2014; 41(10). DOI:10.1007/s11033-014-3550-6 · 1.96 Impact Factor