scholarly journals Structural and functional characterization of buffalo oviduct-specific glycoprotein (OVGP1) expressed during estrous cycle

2019 ◽  
Vol 39 (12) ◽  
Author(s):  
Suman Choudhary ◽  
Jagadeesh Janjanam ◽  
Sudarshan Kumar ◽  
Jai K. Kaushik ◽  
Ashok K. Mohanty
Keyword(s):  
Estrous Cycle ◽  
Structural Model ◽  
Structural Characteristics ◽  
Functional Characterization ◽  
Carbohydrate Binding ◽  
Evolutionary Analysis ◽  
Binding Assays ◽  
Sugar Binding ◽  
Tim Barrel

Abstract Oviduct-specific glycoprotein (OVGP1) is a high molecular weight chitinase-like protein belonging to GH18 family. It is secreted by non-ciliated epithelial cells of oviduct during estrous cycle providing an essential milieu for fertilization and embryo development. The present study reports the characterization of buffalo OVGP1 through structural modeling, carbohydrate-binding properties and evolutionary analysis. Structural model displayed the typical fold of GH18 family members till the boundary of chitinase-like domain further consisting of a large (β/α)8 TIM barrel sub-domain and a small (α+β) sub-domain. Two critical catalytic residues were found substituted in the catalytic centre (Asp to Phe118, Glu to Leu120) compared with the active chitinase. The carbohydrate-binding groove in TIM barrel was lined with various conserved aromatic residues. Molecular docking with different sugars revealed the involvement of various residues in hydrogen-bonding and non-bonded contacts. Most of the substrate-binding residues were conserved except for a few replacements (Ser13, Lys48, Asp49, Pro50, Asp167, Glu199, Gln272 and Phe275) in comparison with other GH18 members. The residues Trp10, Trp79, Asn80, Gln272, Phe275 and Trp334 were involved in recognition of all six ligands. The α+β sub-domain participated in sugar-binding through Thr270, Gln272, Tyr242 and Phe275. The binding assays revealed significant sugar-binding with purified native and recombinant OVGP1. Phylogenetic analysis revealed that OVGP1 was closely related to AMCases followed by other CLPs and evolution of OVGP1 occurred through several gene duplications. This is the first study describing the structural characteristics of OVGP1 that will further help to understand its interaction with gametes to perform crucial reproductive functions.

scholarly journals The rare sugar N-acetylated viosamine is a major component of Mimivirus fibers

2017 ◽  
Vol 292 (18) ◽  
pp. 7385-7394 ◽  
Author(s):  
Francesco Piacente ◽  
Cristina De Castro ◽  
Sandra Jeudy ◽  
Matteo Gaglianone ◽  
Maria Elena Laugieri ◽  
...  
Keyword(s):  
Structural Model ◽  
Functional Characterization ◽  
Virus Genome ◽  
Amino Group ◽  
Rare Sugar ◽  
Functional Annotations ◽  
Terminal Domain ◽  
Icosahedral Capsid

The giant virus Mimivirus encodes an autonomous glycosylation system that is thought to be responsible for the formation of complex and unusual glycans composing the fibers surrounding its icosahedral capsid, including the dideoxyhexose viosamine. Previous studies have identified a gene cluster in the virus genome, encoding enzymes involved in nucleotide-sugar production and glycan formation, but the functional characterization of these enzymes and the full identification of the glycans found in viral fibers remain incomplete. Because viosamine is typically found in acylated forms, we suspected that one of the genes might encode an acyltransferase, providing directions to our functional annotations. Bioinformatic analyses indicated that the L142 protein contains an N-terminal acyltransferase domain and a predicted C-terminal glycosyltransferase. Sequence analysis of the structural model of the L142 N-terminal domain indicated significant homology with some characterized sugar acetyltransferases that modify the C-4 amino group in the bacillosamine or perosamine biosynthetic pathways. Using mass spectrometry and NMR analyses, we confirmed that the L142 N-terminal domain is a sugar acetyltransferase, catalyzing the transfer of an acetyl moiety from acetyl-CoA to the C-4 amino group of UDP-d-viosamine. The presence of acetylated viosamine in vivo has also been confirmed on the glycosylated viral fibers, using GC-MS and NMR. This study represents the first report of a virally encoded sugar acetyltransferase.

scholarly journals Genome-Wide Identification and Expression Analysis of Sucrose Nonfermenting-1-Related Protein Kinase (SnRK) Genes in Triticum aestivum in Response to Abiotic Stress

2021 ◽  
Author(s):  
Shefali Mishra ◽  
Pradeep Sharma ◽  
Rajender Singh ◽  
ratan Tiwari ◽  
Gyanendra Pratap Singh
Keyword(s):  
Triticum Aestivum ◽  
Gene Family ◽  
Structural Characteristics ◽  
Phylogenetic Analyses ◽  
Expression Patterns ◽  
Functional Characterization ◽  
Plant Stress ◽  
Protein Motif ◽  
Genome Wide

The SnRK gene family is a key regulator playing an important role in plant stress response by phosphorylating the target protein to regulate the signalling pathways. The function of SnRK gene family has been reported in many species but is limited to Triticum asetivum. In this study, SnRK gene family in the wheat genome was identified and its structural characteristics were described. One hundred forty-seven SnRK genes distributed across 21 chromosomes were identified in the Triticum aestivum genome and categorised into three subgroups (SnRK1/2/3) based on phylogenetic analyses and domain types. The gene intron-exon structure and protein-motif composition of SnRKs were similar within each subgroup but different amongst the groups. Gene duplication between the wheat, Arabidopsis, rice and barley genomes was also investigated in order to get insight into the evolutionary aspects of the TaSnRK family genes. SnRK genes showed differential expression patterns in leaves, roots, spike, and grains. Redundant stress-related cis-elements were also found in the promoters of 129 SnRK genes and their expression levels varied widely following drought, ABA and light regulated elements. In particular, TaSnRK2.11 had higher and increased expression under the abiotic stresses and can be a candidate gene for the abiotc stress tolerance. The findings will aid in the functional characterization of TaSnRK genes for further research.

scholarly journals Construction of a Novel Chimeric Dextransucrase Fused to the Carbohydrate-Binding Module CBM2a

2021 ◽  
Author(s):  
Reinaldo Fraga Vidal ◽  
Roberto Carlos Aristicas Ribalta ◽  
Lisandra Teresa Martínez Valdés ◽  
Meinardo Lafargue Gámez ◽  
Amanda Montes Alvarez ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Structural Characteristics ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
The Subject ◽  
Encoding Gene ◽  
First Time

The lactic acid bacteria (LAB) have great potential to produce homoexopolysaccharides (HoPS), have been the subject of extensive research efforts, given their health benefits and physicochemical properties. The HoPS functional properties are determined by structural characteristics of varied molecular weights, types of glycosidic linkages, degrees of branching and chemical composition. The dextransucrases (DSases) are responsible of the synthesis of a kind of HoPS (dextran polymers), which are among the first biopolymers produced at industrial scale with applications in medicine and biotechnology. The concept of glycodiversification opens additional applications for DSases. In that sense the design and characterization of new DSases is of prime importance. Previously, we described the isolation and characterization of a novel extracellular dextransucrase (DSR-F) encoding gene. In this study, from DSR-F, we design a novel chimeric dextransucrase DSR-F-∆SP-∆GBD-CBM2a, where DSR-F-∆SP-∆GBD is fused to the carbohydrate-binding module (CBM2a) of the β-1-4 exoglucanase/xylanase Cex (Xyn10A) of Cellulomonas fimi ATCC 484. This dextransucrase variant is active and without alteration in its specificity. The DSR-F-∆SP-∆GBD-CBM2a is purified by cellulose affinity chromatography for the very first time. Our results indicate that new hybrids and chimeric DSases with novel binding capacity to cellulose can be designed to obtain glyco-biocatalysts from renewable lignocellulosic materials.

scholarly journals Functional Characterization of OXYL, A SghC1qDC LacNAc-specific Lectin from The Crinoid Feather Star Anneissia Japonica

Marine Drugs ◽  
2019 ◽  
Vol 17 (2) ◽  
pp. 136 ◽  
Author(s):  
Imtiaj Hasan ◽  
Marco Gerdol ◽  
Yuki Fujii ◽  
Yasuhiro Ozeki
Keyword(s):  
Human Cancer ◽  
Functional Characterization ◽  
Mapk Signaling ◽  
Carbohydrate Binding ◽  
Human Cancer Cell Lines ◽  
Intronless Genes ◽  
Feather Star

We identified a lectin (carbohydrate-binding protein) belonging to the complement 1q(C1q) family in the feather star Anneissia japonica (a crinoid pertaining to the phylum Echinodermata). The combination of Edman degradation and bioinformatics sequence analysis characterized the primary structure of this novel lectin, named OXYL, as a secreted 158 amino acid-long globular head (sgh)C1q domain containing (C1qDC) protein. Comparative genomics analyses revealed that OXYL pertains to a family of intronless genes found with several paralogous copies in different crinoid species. Immunohistochemistry assays identified the tissues surrounding coelomic cavities and the arms as the main sites of production of OXYL. Glycan array confirmed that this lectin could quantitatively bind to type-2 N-acetyllactosamine (LacNAc: Galβ1-4GlcNAc), but not to type-1 LacNAc (Galβ1-3GlcNAc). Although OXYL displayed agglutinating activity towards Pseudomonas aeruginosa, it had no effect on bacterial growth. On the other hand, it showed a significant anti-biofilm activity. We provide evidence that OXYL can adhere to the surface of human cancer cell lines BT-474, MCF-7, and T47D, with no cytotoxic effect. In BT-474 cells, OXYL led to a moderate activation of the p38 kinase in the MAPK signaling pathway, without affecting the activity of caspase-3. Bacterial agglutination, anti-biofilm activity, cell adhesion, and p38 activation were all suppressed by co-presence of LacNAc. This is the first report on a type-2 LacNAc-specific lectin characterized by a C1q structural fold.

scholarly journals Functional Characterization of Carbohydrate-Binding Modules in a New Alginate Lyase, TsAly7B, from Thalassomonas sp. LD5

Marine Drugs ◽  
2019 ◽  
Vol 18 (1) ◽  
pp. 25 ◽  
Author(s):  
Zhelun Zhang ◽  
Luyao Tang ◽  
Mengmeng Bao ◽  
Zhigang Liu ◽  
Wengong Yu ◽  
...  
Keyword(s):  
Specific Activity ◽  
Biological Activities ◽  
Functional Characterization ◽  
Alginate Lyase ◽  
Carbohydrate Binding ◽  
Enzyme Degradation ◽  
Carbohydrate Binding Modules ◽  
Catalytic Module ◽  
Alginate Lyases

Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

scholarly journals Purification and Functional Characterization of the C-Terminal Domain of the β-Actin-Binding Protein AIM1 In Vitro

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3281 ◽  
Author(s):  
Fang Wu ◽  
Liangkai Cheng ◽  
Qi Yu ◽  
Lin Zhang ◽  
Hong Li ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Structural Model ◽  
Functional Characterization ◽  
Actin Binding ◽  
Migration And Invasion ◽  
Binding Modes ◽  
C Terminus ◽  
Biology Research

The protein absent in melanoma 1 (AIM1) is a member of the βγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to β-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the β-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and β-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and β-actin; we also obtain the crystal of the first two βγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the βγ-crystallin domain repeats that bound to β-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research.

scholarly journals Crystal Structure of BRP39 protein expressed during Mammary Gland Apoptosis

2014 ◽  
Vol 70 (a1) ◽  
pp. C1638-C1638
Author(s):  
Suman Choudhary ◽  
Ashok Mohanty ◽  
Andrew Fisher
Keyword(s):  
Breast Cancer ◽  
Crystal Structure ◽  
Carbohydrate Binding ◽  
Potential Candidate ◽  
Structure Based Drug Design ◽  
Sugar Binding ◽  
Replacement Method ◽  
Tim Barrel ◽  
Molecular Replacement Method ◽  
Binding Groove

Breast regression protein (BRP39) from mice is a 39 kDa protein which is a member of chitolectin class of GH18 family. High levels of expression of BRP39 have been detected in breast carcinoma. It has been proposed that this may act as a protecting signalling factor and also help in proliferation of cells during breast cancer. It can act as a potential candidate for rational structure based drug design against breast cancer. Here, we report the crystal structure of recombinant BRP39 in a deglycosylated form which was expressed in a heterologous system i.e E.Coli. To understand the role of sugar moiety, crystal structure of a deglycosylated chitolectin is an essential requirement. The structure was solved by molecular replacement method of phase determination and refined to a 2.6 A0resolution. The overall structure of BRP39 consists of two globular domains: a large (β/α)8 TIM barrel domain and a small (α+β) domain. The most striking observation from BRP39 structure is the conformation of critical Trp 100 residue into the β-barrel of carbohydrate binding groove of BRP39. The structure reveals that the glycan moiety plays an important role in the orientation of critical Trp100 in the β-barrel. In deglycosylated BRP39, it orients away from the barrel and resembles the conformation as seen in non-glycosylated chitinases. In contrast to this, the corresponding Trp is oriented into the barrel in case of other glycosylated homologues of BRP39 which may have its implications in sugar binding. Furthermore, in MGP-40, the altered conformation of loop is stabilized by H-bonding and stacking interactions whereas in BRP39, no hydrogen bond was observed between any of the residues of this loop except for Trp100 which interacts with a water molecule may be to stabilize its conformation. Another important observation in the sugar binding groove of BRP39 structure is the mutation of two important residues, one in TIM barrel domain and another in a part of α+β domain which is also involved in sugar binding. Asn100 and Arg263 in Hcgp39 and other chitinase- like proteins (SPX-40 structures) are replaced by Lys101 and Lys264 in BRP39. Both of these residues i.e. Asn100 and Arg 263 in HCgp39 have been reported to be involved in stabilizing the binding of GlcNAc inside the groove. Possibly, there is a significant distortion in the shape of sugar binding groove due to these mutations in BRP39.

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