Evidence for the participation of -hexosaminidase in human sperm-zona pellucida interaction in vitro

Zhang

Journal of Biological Chemistry

et al. 2011

119

␤-N-Acetyl-D-hexosaminidase has been postulated to have a specialized function. However, the structural basis of this specialization is not yet established. OfHex1, the enzyme from the Asian corn borer Ostrinia furnacalis (one of the most destructive pests) has previously been reported to function merely in chitin degradation. Here the vital role of OfHex1 during the pupation of O. furnacalis was revealed by RNA interference, and the crystal structures of OfHex1 and OfHex1 complexed with TMG-chitotriomycin were determined at 2.1 Å . The mechanism of selective inhibition by TMG-chitotriomycin was related to the existence of the ؉1 subsite at the active pocket of OfHex1 and a key residue, Trp 490 , at this site. Mutation of Trp 490 to Ala led to a 2,277-fold decrease in sensitivity toward TMG-chitotriomycin as well as an 18-fold decrease in binding affinity for the substrate (GlcNAc) 2 . Although the overall topology of the catalytic domain of OfHex1 shows a high similarity with the human and bacterial enzymes, OfHex1 is distinguished from these enzymes by large conformational changes linked to an "open-close" mechanism at the entrance of the active site, which is characterized by the "lid" residue, Trp 448 . Mutation of Trp 448 to Ala or Phe resulted in a more than 1,000-fold loss in enzyme activity, due mainly to the effect on k cat . The current work has increased our understanding of the structure-function relationship of OfHex1, shedding light on the structural basis that accounts for the specialized function of ␤-N-acetyl-D-hexosaminidase as well as making the development of species-specific pesticides a likely reality.␤-N-Acetyl-D-hexosaminidase (EC 3.2.1.52), a member of the family 20 glycosyl hydrolyases (GH20), 4 is an enzyme that participates in the breakdown of glycosidic bonds of glycans, glycoproteins, and glycolipids (1). It has been postulated to have specialized physiological functions, including post-translational modification of N-glycans, degradation of glycoconjugates, and egg-sperm recognition (1). The structural basis for these specialized functions is still unclear.It is interesting to note that insects have evolved to have more than one ␤-N-acetyl-D-hexosaminidase, as revealed by genomic analysis of various insects, including Coleoptera, Diptera, Hymenoptera, Lepidoptera, Phthiraptera, and Hemiptera. The activities of insect ␤-N-acetyl-D-hexosaminidases are not restricted to chitin degradation but are also associated with post-translational modification of N-glycans, degradation of glycoconjugates, and egg-sperm recognition, suggesting that these enzymes have rather versatile physiological functions in the growth and development of insects (2). Some of these physiological functions may overlap with those of the same enzymes found in higher organisms. Mammal lysosomal ␤-N-acetyl-D-hexosaminidases are mainly responsible for glycoconjugate degradation in lysosome (3). Likewise, ␤-N-acetyl-D-hexosaminidases from the insects Bombyx mori (4) and Spodoptera frugiperda (5) have broad substrate spe...

mentioning

confidence: 99%

Structural Determinants of an Insect β-N-Acetyl-d-hexosaminidase Specialized as a Chitinolytic Enzyme

Zhang

Journal of Biological Chemistry

et al. 2011

119

Journal of Reproduction and Development

“…Oligosaccharides on the ZP include some monosaccharides, such as N-acetyl-D-glucosamine (GlcNAc), D-mannose and D-fucose [2], and these monosaccharides are involved in many sperm-oocyte interactions. For example, in sperm-oocyte recognition, N-acetyl-D-glucosaminidase (NAG) inhibits sperm binding to the ZP in the human [3,4]. The mouse sperm has 20-fold more NAG compared with other enzymes [5], and recognition of GlcNAc on the ZP glycoprotein ZP3 by β1-4-galactosyltransferase on the sperm acrosome membrane is dependent on the initial attachment of the sperm to the ZP [6,7].…”

mentioning

confidence: 99%

Effect of N-Acetyl-D-Glucosamine on Bovine Sperm-Oocyte Interactions

Sakaguchi

Iwata

Kuwayama

et al. 2009

Abstract. N-Acetyl-D-glucosamine (GlcNAc) is a major component of glycosaminoglycan, which is involved in spermoocyte interactions. We examined the effect of adding GlcNAc and other monosaccharides, D-mannose and D-fucose, to the in vitro fertilization (IVF) medium on bovine sperm-oocyte interactions. In medium in which sperm and a zona pellucida (ZP) were co-incubated with monosaccharides for 5 min, addition of GlcNAc (5 or 25 mM) significantly reduced the number of sperm that attached to the ZP. Pretreatment of gametes with GlcNAc (5 mM) prior to coincubation also suppressed sperm-ZP attachment. Addition of GlcNAc (5 or 25 mM) to the medium in which sperm and a ZP were co-incubated for 5 h, however, significantly increased the number of sperm binding to and penetrating the ZP in a concentration-related manner. The other monosaccharides, D-fucose and D-mannose, did not have this effect. Supplementation of the sperm-oocyte co-incubation medium with 5 mM GlcNAc also enhanced the rate of polyspermic fertilization. When the ZPs were removed from the oocytes, GlcNAc did not affect the fertilization rate. Furthermore, incubation of sperm with 5 mM GlcNAc induced sperm membrane destabilization and an acrosome reaction, as evidenced by the hypo-osmotic swelling test and fluorescein isothiocyanate-labeled peanut agglutinin/ propidium iodide (FITC-PNA/PI) staining. Finally, GlcNAc suppressed ZP hardening following fertilization, as determined by measuring the time required for pronase to dissolve the ZP. In conclusion, supplementation of IVF medium with GlcNAc has various effects on sperm-oocyte interactions including suppression of initial attachment, induction of sperm membrane destabilization and acrosome reaction, increase in the number of sperm secondarily bound to and penetrating the ZP, suppression of ZP hardening following sperm-oocyte co-incubation and increase in the rate of polyspermic fertilization. Key words: Bovine, Fertilization, Monosaccharides, Sperm, Zona pellucida (J. Reprod. Dev. 55: [676][677][678][679][680][681][682][683][684] 2009) ertilization of oocytes comprises several interactions between the sperm and oocyte. Contact between the sperm and oocyte first occurs at the outer surface of the surrounding extracellular matrix of the egg, the zona pellucida (ZP). Binding of the lectinlike protein on the sperm head to distinct oligosaccharides of ZP glycoproteins leads the capacitated sperm to recognize the oocyte. Upon anchoring of the sperm to the oocyte through the ZP carbohydrates, a signaling cascade induces the acrosome reaction of the sperm, allowing the sperm to pass through the ZP. The acrosomereacted, penetrated sperm fuses with the egg plasma membrane, which enables the egg to complete meiosis, and initiates mechanisms to prevent polyspermic fertilization by the exocytosis of cortical granules (CG) [1]. Oligosaccharides on the ZP include some monosaccharides, such as N-acetyl-D-glucosamine (GlcNAc), D-mannose and D-fucose [2], and these monosaccharides are involved in many sperm-oocyte in...

“…Glycosidases mostly come from the epididymis, where they modify sperm surface glycoproteins during maturation (Dacheux et al 2006). Additionally, some glycosidases bind to sperm membrane glycosidic residues and act as sites to either recognize the oviductal epithelium (Talevi & Gualtieri 2010) or to participate in human sperm-egg interaction through zona-pellucida glycoproteins (Miranda et al 2000). Hexosaminidases bind to human sperm membrane during epididymal transit (Perez Martinez et al 2008) and participate in sperm-zona pellucida interaction (Miranda et al 2000).…”

Section: Proteins Mediating Formation Of the Oviduct Reservoir And Spmentioning

confidence: 99%

“…Additionally, some glycosidases bind to sperm membrane glycosidic residues and act as sites to either recognize the oviductal epithelium (Talevi & Gualtieri 2010) or to participate in human sperm-egg interaction through zona-pellucida glycoproteins (Miranda et al 2000). Hexosaminidases bind to human sperm membrane during epididymal transit (Perez Martinez et al 2008) and participate in sperm-zona pellucida interaction (Miranda et al 2000). In the peccary seminal fluid, hexosaminidase is expressed as four isoforms with 102 kDa, very similar to the boar seminal hexosaminidase (Daron & Aull 1985).…”

Section: Proteins Mediating Formation Of the Oviduct Reservoir And Spmentioning

confidence: 99%

Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758)

Santos¹,

Sousa²,

Martins³

et al. 2014

Reproduction

This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC-MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2G6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7G8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (rZK0.87; P!0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4G1.9 and 8.8G3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-a-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.