Carving out a Glycoside Hydrolase Active Site for Incorporation into a New Protein Scaffold Using Deep Network Hallucination.

Enzymes are indispensable biocatalysts for numerous industrial applications, yet stability, selectivity, and restricted substrate recognition present limitations for their use. Despite the importance of enzyme engineering in overcoming these limitations, success is often challenged by the intricate architecture of enzymes derived from natural sources. Recent advances in computational methods have enabled the de novo design of simplified scaffolds with specific functional sites. Such scaffolds may be advantageous as platforms for enzyme engineering. Here, we present a strategy for the de novo design of a simplified scaffold of an endo-α-N-acetylgalactosaminidase active site, a glycoside hydrolase from the GH101 enzyme family. Using a combination of trRosetta hallucination, iterative cycles of deep-learning-based structure prediction, and ProteinMPNN sequence design, we designed proteins with 290 amino acids incorporating the active site while reducing the molecular weight by over 100 kDa compared to the initial endo-α-N-acetylgalactosaminidase. Of 11 tested designs, six were expressed as soluble monomers, displaying similar or increased thermostabilities compared to the natural enzyme. Despite lacking detectable enzymatic activity, the experimentally determined crystal structures of a representative design closely matched the design with a root-mean-square deviation of 1.0 Å, with most catalytically important side chains within 2.0 Å. The results highlight the potential of scaffold hallucination in designing proteins that may serve as a foundation for subsequent enzyme engineering.

Silencing of α-N-acetylgalactosaminidase in the gastric cancer cells amplified cell death and attenuated migration, while the multidrug resistance remained unchanged.

Although the elevated level of the α-N-acetylgalactosaminidase enzyme (encoded by the NAGA gene) is a well-recognized feature of cancer cells; little research works have been undertaken on the cancer malignancy mechanisms. The effects of NAGA gene downregulation on cancer cells' features such as drug resistance, impaired programmed cell death, and migration were analyzed in this study. The cells grew exponentially with a doubling time of 30 h in an optimal condition. Toxicity of daunorubicin chemotherapy drug on NAGA-transfected EPG85.257RDB cells was evaluated in comparison to control cells and no significant change was recorded. Quantitative transcript analyses and protein levels revealed that the MDR1 pump almost remained unchanged during the study. Moreover, the NAGA gene downregulation enhanced the late apoptosis rate in EPG85.257RDB cells at 24 h posttransfection. The investigated expression level of genes and proteins involved in the TNFR2 signaling pathway, related to cancer cell apoptosis, showed considerable alterations after NAGA silencing as well. MAP3K14 and CASP3 genes were downregulated while IL6, RELA, and TRAF2 experienced an upregulation. Also, NAGA silencing generally diminished the migration ability of EPG85.257RDB cells and the MMP1 gene (as a critical gene in metastasis) expression decreased significantly. The expression of the p-FAK protein, which is located in the downstream of the α2 ß1 integrin signaling pathway, was reduced likewise. It could be concluded that despite drug resistance, NAGA silencing resulted in augmentative and regressive effects on cell death and migration.

Structural and mechanistic insights into the substrate specificity and hydrolysis of GH31 α-N-acetylgalactosaminidase.

Glycoside hydrolase family 31 (GH31) is a diversified family of anomer-retaining α-glycoside hydrolases, such as α-glucosidase and α-xylosidase, among others. Recently, GH31 α-N-acetylgalactosaminidases (Nag31s) have been identified to hydrolyze the core of mucin-type O-glycans and the crystal structure of a gut bacterium Enterococcus faecalis Nag31 has been reported. However, the mechanisms of substrate specificity and hydrolysis of Nag31s are not well investigated. Herein, we show that E. faecalis Nag31 has the ability to release N-acetylgalactosamine (GalNAc) from O-glycoproteins, such as fetuin and mucin, but has low activity against Tn antigen. Mutational analysis and crystal structures of the Michaelis complexes reveal that residues of the active site work in concert with their conformational changes to act on only α-N-acetylgalactosaminides. Docking simulations using GalNAc-attached peptides suggest that the enzyme mainly recognizes GalNAc and side chains of Ser/Thr, but not strictly other peptide residues. Moreover, quantum mechanics calculations indicate that the enzyme preferred p-nitrophenyl α-N-acetylgalactosaminide to Tn antigen and that the hydrolysis progresses through a conformational itinerary, 4C1 → 1S3 → 4C1, in GalNAc of substrates. Our results provide novel insights into the diversification of the sugar recognition and hydrolytic mechanisms of GH31 enzymes.

Identification of a Catalytic Nucleophile-Activating Network in the endo-α-N-Acetylgalactosaminidase of Family GH101.

Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galß1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galß(1-3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile.

A missense variant in NDUFA6 confers schizophrenia risk by affecting YY1 binding and NAGA expression.

Genome-wide association studies (GWASs) have revealed that genetic variants at the 22q13.2 risk locus were robustly associated with schizophrenia. However, the causal variants at this risk locus and their roles in schizophrenia remain elusive. Here we identify the risk missense variant rs1801311 (located in the 1st exon of NDUFA6 gene) as likely causal for schizophrenia at 22q13.2 by disrupting binding of YY1, TAF1, and POLR2A. We systematically elucidated the regulatory mechanisms of rs1801311 and validated the regulatory effect of this missense variant. Intriguingly, rs1801311 physically interacted with NAGA (encodes the alpha-N-acetylgalactosaminidase, which is mainly involved in regulating metabolisms of glycoproteins and glycolipids in lysosome) and showed the most significant association with NAGA expression in the human brain, with the risk allele (G) associated with higher NAGA expression. Consistent with eQTL analysis, expression analysis showed that NAGA was significantly upregulated in brains of schizophrenia cases compared with controls, further supporting that rs1801311 may confer schizophrenia risk by regulating NAGA expression. Of note, we found that NAGA regulates important neurodevelopmental processes, including proliferation and differentiation of neural stem cells. Transcriptome analysis corroborated that NAGA regulates pathways associated with neuronal differentiation. Finally, we independently confirmed the association between rs1801311 and schizophrenia in a large Chinese cohort. Our study elucidates the regulatory mechanisms of the missense schizophrenia risk variant rs1801311 and provides mechanistic links between risk variant and schizophrenia etiology. In addition, this study also revealed the novel role of coding variants in gene regulation and schizophrenia risk, i.e., genetic variant in coding region of a specific gene may confer disease risk through regulating distal genes (act as regulatory variant for distal genes).

Biochemical characterization of Bombyx mori α-N-acetylgalactosaminidase belonging to the glycoside hydrolase family 31.

Horizontal gene transfer is an important evolutionary mechanism not only for bacteria but also for eukaryotes. In the domestic silkworm Bombyx mori, a model species of lepidopteran insects, some enzymes are known to have been acquired by horizontal transfer; however, the enzymatic features of protein BmNag31, belonging to glycoside hydrolase family 31 (GH31) and whose gene was predicted to be transferred from Enterococcus sp. are unknown. In this study, we reveal that the transcription of BmNag31 increases significantly during the prepupal to pupal stage, and decreases in the adult stage. The full-length BmNag31 and its truncated mutants were heterologously expressed in Escherichia coli and characterized. Its catalytic domain exhibits α-N-acetylgalactosaminidase activity and the carbohydrate-binding module family 32 domain shows binding activity towards N-acetylgalactosamine, similar to the Enterococcus faecalis homolog, EfNag31A. Gel filtration chromatography and blue native polyacrylamide gel electrophoresis analyses indicate that BmNag31 forms a hexamer whereas EfNag31A is monomeric. These results provide insights into the function of lepidopteran GH31 α-N-acetylgalactosaminidase.

Synthesis and evaluation of sensitive coumarin-based fluorogenic substrates for discovery of α-N-acetyl galactosaminidases through droplet-based screening.

As part of a search for a substrate for droplet-based microfluidic screening assay of α-N-acetylgalactosaminidases, spectral and physical characteristics of a series of coumarin derivatives were measured. From among these a new coumarin-based fluorophore, Jericho Blue, was selected as having optimal characteristics for our screen. A reliable method for the challenging synthesis of coumarin glycosides of α-GalNAc was then developed and demonstrated with nine examples. The α-GalNAc glycoside of Jericho Blue prepared in this way was shown to function well under screening conditions.

Biochemical characterization of the endo-α-N-acetylgalactosaminidase pool of the human gut symbiont Tyzzerella nexilis.

Three large (2084-, 984-, and 2104-amino acids) endo-α-N-acetylgalactosaminidase candidate genes from the commensal human gut bacterium Tyzzerella nexilis were successfully cloned and subsequently expressed in Escherichia coli. Activity tests of the purified proteins revealed that two of the candidate genes (Tn0153 and Tn2105) were able to hydrolyze the disaccharide unit from Galß1-3GalNAc-α-pNP. The biochemical characterization revealed optimum pH conditions of 4.0 for both enzymes and temperature optima of 50 °C. The addition of 2-mercaptoethanol, Triton X-100 and urea had only minor effects on the activity of the enzymes, and the addition of imidazole and sodium dodecyl sulfate led to a significant reduction of the enzymes' activities. A mutational study identified and confirmed the role of the catalytically significant amino acids. The present study describes the first functional characterization of members of the GH101 family from this human gut symbiont.

The EngCP endo α-N-acetylgalactosaminidase is a virulence factor involved in Clostridium perfringens gas gangrene infections.

Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.

A Novel Homozygous Missense Variant in the NAGA Gene with Extreme Intrafamilial Phenotypic Heterogeneity.

Schindler disease is a rare autosomal recessive lysosomal storage disorder caused by a deficiency in alpha-N-acetylgalactosaminidase (α-NAGA) activity due to defects in the NAGA gene. Accumulation of the enzyme's substrates results in clinically heterogeneous symptoms ranging from asymptomatic individuals to individuals with severe neurological manifestations. Here, a 5-year-old Emirati male born to consanguineous parents presented with congenital microcephaly and severe neurological manifestations. Whole genome sequencing revealed a homozygous missense variant (c.838C>A; p.L280I) in the NAGA gene. The allele is a reported SNP in the ExAC database with a 0.0007497 allele frequency. The proband's asymptomatic sister and cousin carry the same genotype in a homozygous state as revealed from the family screening. Due to the extreme intrafamilial heterogeneity of the disease as seen in previously reported cases, we performed further analyses to establish the pathogenicity of this variant. Both the proband and his sister showed abnormal urine oligosaccharide patterns, which is consistent with the diagnosis of Schindler disease. The α-NAGA activity was significantly reduced in the proband and his sister with 5.9% and 12.1% of the mean normal activity, respectively. Despite the activity loss, p.L280I α-NAGA processing and trafficking were not affected. However, protein molecular dynamic simulation analysis revealed that this amino acid substitution is likely to affect the enzyme's natural dynamics and hinders its ability to bind to the active site. Functional analysis confirmed the pathogenicity of the identified missense variant and the diagnosis of Schindler disease. Extreme intrafamilial clinical heterogeneity of the disease necessitates further studies for proper genetic counseling and management.

Prospecting for microbial α-N-acetylgalactosaminidases yields a new class of GH31 O-glycanase.

α-Linked GalNAc (α-GalNAc) is most notably found at the nonreducing terminus of the blood type-determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans. However, despite their ubiquity in saccharolytic microbe-rich environments such as the human gut, relatively few α-N-acetylgalactosaminidases are known. Here, to discover and characterize novel microbial enzymes that hydrolyze α-GalNAc, we screened small-insert libraries containing metagenomic DNA from the human gut microbiome. Using a simple fluorogenic glycoside substrate, we identified and characterized a glycoside hydrolase 109 (GH109) that is active on blood type A-antigen, along with a new subfamily of glycoside hydrolase 31 (GH31) that specifically cleaves the initial α-GalNAc from mucin-type O-glycans. This represents a new activity in this GH family and a potentially useful new enzyme class for analysis or modification of O-glycans on protein or cell surfaces.

cPLA2 activation contributes to lysosomal defects leading to impairment of autophagy after spinal cord injury.

The autophagy-lysosomal pathway plays an essential role in cellular homeostasis as well as a protective function against a variety of diseases including neurodegeneration. Conversely, inhibition of autophagy, for example due to lysosomal dysfunction, can lead to pathological accumulation of dysfunctional autophagosomes and consequent neuronal cell death. We previously reported that autophagy is inhibited and contributes to neuronal cell death following spinal cord injury (SCI). In this study, we examined lysosomal function and explored the mechanism of lysosomal defects following SCI. Our data demonstrated that expression levels and processing of the lysosomal enzyme cathepsin D (CTSD) are decreased by 2 h after SCI. Enzymatic activity levels of CTSD and another lysosomal enzyme, N-acetyl-alpha-glucosaminidase, are both decreased 24 h post injury, indicating general lysosomal dysfunction. Subcellular fractionation and immunohistochemistry analysis demonstrated that this dysfunction is due to lysosomal membrane permeabilization and leakage of lysosomal contents into the cytosol. To directly assess extent and mechanisms of damage to lysosomal membranes, we performed mass spectrometry-based lipidomic analysis of lysosomes purified from SCI and control spinal cord. At 2 h post injury our data demonstrated increase in several classes of lysosophospholipids, the products of phospholipases (PLAs), as well as accumulation of PLA activators, ceramides. Phospholipase cPLA2, the main PLA species expressed in the CNS, has been previously implicated in mediation of secondary injury after SCI, but the mechanisms of its involvement remain unclear. Our data demonstrate that cPLA2 is activated within 2 h after SCI preferentially in the lysosomal fraction, where it colocalizes with lysosomal-associated membrane protein 2 in neurons. Inhibition of cPLA2 in vivo decreased lysosomal damage, restored autophagy flux, and reduced neuronal cell damage. Taken together our data implicate lysosomal defects in pathophysiology of SCI and for the first time indicate that cPLA2 activation leads to lysosomal damage causing neuronal autophagosome accumulation associated with neuronal cell death.

Steady state kinetic analysis of O-linked GalNAc glycan release catalyzed by endo-α-N-acetylgalactosaminidase.

Often glycosidase assays are based on small-molecule compounds where a glycan of interest is linked to a chromophore allowing for easy detection of cleavage of the glycoside bond. However, such compounds only resemble part of the more complex substrate molecule for enzymes acting on glycoconjugates of glycopeptides or glycoproteins. Nonetheless, the advantage is obvious as enzyme activity is readily recorded and kinetic parameters easily obtained. This is not often the case with glycopeptides or glycoproteins as these may reveal increased complexity in terms of heterogeneity in protein-glycan stoichiometry and restricted enzyme accessibility. However, a kinetic analysis of glycan release from glycopeptides could provide information complementary to that of small-molecule substrates, especially if providing kinetic parameters that are immediately comparable. We have characterized the steady state kinetics of wild type and mutant variants of Bifidobacterium longum endo-α-N-acetylgalactosaminidase, by recording the enzymatic release of Galß(1-3)GalNAc from bovine glycomacropeptide pre-treated with sialidase to remove sialic acid units. Differences between previously reported kinetic constants obtained with synthetic substrates and those obtained in the present work demonstrate an influence of the peptide moiety on the kinetic properties of endo-α-N-acetylgalactosaminidase. The devised assay and data handling method determines the accessible substrate concentration as well as the steady state kinetic parameters, KM and kcat, for glycoconjugates of glycopeptides described by the same units as obtained from using small-molecule substrates and thus allows for a direct comparison.

Functional and solution structure studies of amino sugar deacetylase and deaminase enzymes from Staphylococcus aureus.

N-Acetylglucosamine-6-phosphate deacetylase (NagA) and glucosamine-6-phosphate deaminase (NagB) are branch point enzymes that direct amino sugars into different pathways. For Staphylococcus aureus NagA, analytical ultracentrifugation and small-angle X-ray scattering data demonstrate that it is an asymmetric dimer in solution. Initial rate experiments show hysteresis, which may be related to pathway regulation, and kinetic parameters similar to other bacterial isozymes. The enzyme binds two Zn2+ ions and is not substrate inhibited, unlike the Escherichia coli isozyme. S. aureus NagB adopts a novel dimeric structure in solution and shows kinetic parameters comparable to other Gram-positive isozymes. In summary, these functional data and solution structures are of use for understanding amino sugar metabolism in S. aureus, and will inform the design of inhibitory molecules.

Carotenoids produced by yeast biomass protect mechanisms regulating endothelial barrier function from lipopolysaccharide-induced damage in the rat heart.

Expression of occludin and junctional adhesion molecule A (JAM-A), transmembrane proteins of tight junctions (TJs), was analysed to characterize endothelial paracellular permeability in the heart of rats subjected to a bolus of bacterial lipopolysaccharide (LPS) at a dose of 1 mg/kg. Potential protective effects of natural carotenoids (10 mg/kg/day) produced by yeast biomass Rhodosporidium kratochvilovae on expression of occludin and JAM-A also examined in LPS-injected rats (n = 6 per group). LPS decreased expression of occludin by 40% (P < 0.01), JAM-A by 36% (P < 0.001) and increased plasma levels of malondialdehyde (MDA) and lysosomal N-acetyl-b-D-glucosaminidase (NAGA) compared to controls. Ten-day diet rich in yeast biomass containing carotenoids (torularhodin, torulene, ß-carotene) attenuated LPS-induced changes in expression of TJ proteins as observed by increased expression of occludin by 30% (P < 0.05) and JAM-A by 61% (P < 0.001) to the control values. Carotenoids also reduced oxidative stress and cellular injury indicated by decreased levels of MDA and NAGA. The results show that diet rich in yeast biomass containing natural carotenoids could protect mechanisms regulating paracellular endothelial barrier function from LPS-induced damage in the heart.

Carbohydrate Syntrophy enhances the establishment of Bifidobacterium breve UCC2003 in the neonatal gut.

The non-digestible oligosaccharide fraction of maternal milk represents an important of carbohydrate and energy source for saccharolytic bifidobacteria in the gastrointestinal tract during early life. However, not all neonatal bifidobacteria isolates can directly metabolise the complex sialylated, fucosylated, sulphated and/or N-acetylglucosamine-containing oligosaccharide structures present in mothers milk. For some bifidobacterial strains, efficient carbohydrate syntrophy or crossfeeding is key to their establishment in the gut. In this study, we have adopted advanced functional genomic approaches to create single and double in-frame deletions of the N-acetyl glucosamine 6-phosphate deacetylase encoding genes, nagA1 and nagA2, of B. breve UCC2003. In vitro phenotypic analysis followed by in vivo studies on co-colonisation, mother to infant transmission, and evaluation of the relative co-establishment of B. bifidum and B. breve UCC2003 or UCC2003ΔnagA1ΔnagA2 in dam-reared neonatal mice demonstrates the importance of crossfeeding on sialic acid, fucose and N-acetylglucosamine-containing oligosaccharides for the establishment of B. breve UCC2003 in the neonatal gut. Furthermore, transcriptomic analysis of in vivo gene expression shows upregulation of genes associated with the utilisation of lactose, sialic acid, GlcNAc-6-S and fucose in B. breve UCC2003, while for UCC2003ΔnagA1ΔnagA2 only genes for lactose metabolism were upregulated.

The Effect of Fucoidan from the Brown Alga Fucus evanescence on the Activity of α-N-Acetylgalactosaminidase of Human Colon Carcinoma Cells.

α-N-acetylgalactosaminidase (EC 3.2.1.49) (alpha-NaGalase) catalyzes the hydrolysis of N-acetamido-2-deoxy-α-d-galactoside residues from non-reducing ends of various complex carbohydrates and glycoconjugates. It is known that human cancer cells express an alpha-NaGalase, which accumulates in the blood plasma of patients. The enzyme deglycosylates the Gc protein-derived macrophage activating factor (GcMAF) and inhibits macrophage activity acting as an immunosuppressor. The high specific activity 0.033 ± 0.002 μmol mg−1 min−1 of the enzyme was found in human colon carcinoma cells DLD-1. The alpha-NaGalase of DLD-1 cells was isolated and biochemical characterized. The enzyme exhibits maximum activity at pH 5.2 and temperature 55 °C. The Km is 2.15 mM, Vmax⁻0.021 μmol min−1 mL−1, kcat⁻1.55 min−1 and kcat/Km⁻0.72 min−1 mM−1 at 37 °C, pH 5.2. The effects of fucoidan from the brown alga Fucus evanescence on the activity of alpha-NaGalase in human colon carcinoma DLD-1 cells and on the biosynthesis of this enzyme were investigated. It was shown that fucoidan did not inhibit free alpha-NaGalase, however, it reduced the expression of the enzyme in the DLD-1 cells at IC50 73 ± 4 μg mL−1.

The first crystal structure of a family 129 glycoside hydrolase from a probiotic bacterium reveals critical residues and metal cofactors.

The α-N-acetylgalactosaminidase from the probiotic bacterium Bifidobacterium bifidum (NagBb) belongs to the glycoside hydrolase family 129 and hydrolyzes the glycosidic bond of Tn-antigen (GalNAcα1-Ser/Thr). NagBb is involved in assimilation of O-glycans on mucin glycoproteins by B. bifidum in the human gastrointestinal tract, but its catalytic mechanism has remained elusive because of a lack of sequence homology around putative catalytic residues and of other structural information. Here we report the X-ray crystal structure of NagBb, representing the first GH129 family structure, solved by the single-wavelength anomalous dispersion method based on sulfur atoms of the native protein. We determined ligand-free, GalNAc, and inhibitor complex forms of NagBb and found that Asp-435 and Glu-478 are located in the catalytic domain at appropriate positions for direct nucleophilic attack at the anomeric carbon and proton donation for the glycosidic bond oxygen, respectively. A highly conserved Asp-330 forms a hydrogen bond with the O4 hydroxyl of GalNAc in the -1 subsite, and Trp-398 provides a stacking platform for the GalNAc pyranose ring. Interestingly, a metal ion, presumably Ca2+, is involved in the recognition of the GalNAc N-acetyl group. Mutations at Asp-435, Glu-478, Asp-330, and Trp-398 and residues involved in metal coordination (including an all-Ala quadruple mutant) significantly reduced the activity, indicating that these residues and the metal ion play important roles in substrate recognition and catalysis. Interestingly, NagBb exhibited some structural similarities to the GH101 endo-α-N-acetylgalactosaminidases, but several critical differences in substrate recognition and reaction mechanism account for the different activities of these two enzymes.

Chemical and structural characterization of α-N-acetylgalactosaminidase I and II from starfish, asterina amurensis.

BACKGROUND: The marine invertebrate starfish was found to contain a novel α-N-acetylgalactosaminidase, α-GalNAcase II, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc), in addition to a typical α-N-acetylgalactosaminidase, α-GalNAcase I, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc) and, to a lesser extent, galactose. The interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their differences in substrate specificity remain unknown. RESULTS: Chemical and structural comparisons between α-GalNAcase I and II using immunostaining, N-terminal amino acid sequencing and peptide analysis showed high homology to each other and also to other glycoside hydrolase family (GHF) 27 members. The amino acid sequence of peptides showed conserved residues at the active site as seen in typical α-GalNAcase. Some substitutions of conserved amino acid residues were found in α-GalNAcase II that were located near catalytic site. Among them G171 and A173, in place of C171 and W173, respectively in α-GalNAcase were identified to be responsible for lacking intrinsic α-galactosidase activity of α-GalNAcase II. Chemical modifications supported the presence of serine, aspartate and tryptophan as active site residues. Two tryptophan residues (W16 and W173) were involved in α-galactosidase activity, and one (W16) of them was involved in α-GalNAcase activity. CONCLUSIONS: The results suggested that α-GalNAcase I and II are closely related with respect to primary and higher order structure and that their structural differences are responsible for difference in substrate specificities.

Identification and characterization of a bovine sperm acrosomal matrix protein and its mechanism of interaction with acrosomal hydrolases.

Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads comprised 54, 50, 45, and 38-19 kDa polypeptides. The objective of this study is to identify and characterize the 45 kDa (OMC45) polypeptide, to define its role in binding acrosomal hydrolases, and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI-TOF-TOF yielded eight peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X-100-permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti-OMC45 and anti-TEKT3. The OMC45 polypeptide was solubilized by radio-immunoprecipitation assay buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti-OMC45 immunoprecipitation pellet. An identical blot stained with anti-TEKT3 exhibited the presence of TEKT3 polypeptide in the anti-OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide and that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N-linked and O-linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N-acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid membrane complex.