Publications by authors named "Saharuddin Bin Mohamad"

20 Publications

  • Page 1 of 1

A single-center pilot study in Malaysia on the clinical utility of whole-exome sequencing for inborn errors of immunity.

Clin Exp Immunol 2021 Jun 1. Epub 2021 Jun 1.

Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

Primary immunodeficiency diseases refer to inborn errors of immunity (IEI) that affect the normal development and function of the immune system. The phenotypical and genetic heterogeneity of IEI have made their diagnosis challenging. Hence, whole-exome sequencing (WES) was employed in this pilot study to identify the genetic etiology of 30 pediatric patients clinically diagnosed with IEI. The potential causative variants identified by WES were validated using Sanger sequencing. Genetic diagnosis was attained in 46.7% (14 of 30) of the patients and categorized into autoinflammatory disorders (n = 3), diseases of immune dysregulation (n = 3), defects in intrinsic and innate immunity (n = 3), predominantly antibody deficiencies (n = 2), combined immunodeficiencies with associated and syndromic features (n = 2) and immunodeficiencies affecting cellular and humoral immunity (n = 1). Of the 15 genetic variants identified, two were novel variants. Genetic findings differed from the provisional clinical diagnoses in seven cases (50.0%). This study showed that WES enhances the capacity to diagnose IEI, allowing more patients to receive appropriate therapy and disease management.
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http://dx.doi.org/10.1111/cei.13626DOI Listing
June 2021

Atypical Presentation of Severe Fungal Necrotizing Fasciitis in a Patient with X-Linked Agammaglobulinemia.

J Clin Immunol 2021 Aug 13;41(6):1178-1186. Epub 2021 Mar 13.

Primary Immunodeficiency Unit, Allergy and Immunology Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health, Selangor, Malaysia.

X-linked agammaglobulinemia is a rare primary immunodeficiency due to a BTK mutation. The patients are characteristically deficient in peripheral B cells and serum immunoglobulins. While they are susceptible to infections caused by bacteria, enteroviruses, and parasites, fungal infections are uncommon in XLA patients. Here, we report a boy of Malay ethnicity who suffered from recurrent upper respiratory tract infections and severe progressive necrotizing fasciitis caused by Saksenaea erythrospora. Immunological tests showed a B cell deficiency and hypogammaglobulinemia. Whole-exome sequencing identified a dinucleotide deletion (c.1580_1581del) in BTK, confirmed by Sanger sequencing and predicted to be disease causing by in silico functional prediction tools (Varsome and MutationTaster2) but was absent in the gnomAD database. This mutation resulted in a frameshift and premature termination (p.C527fs), which disrupted the protein structure. The mother was heterozygous at the mutation site, confirming her carrier status. Flow cytometric analysis of monocyte BTK expression showed it to be absent in the patient and bimodal in the mother. This study describes a novel BTK mutation in a defined hotspot and an atypical fungal phenotype in XLA. Further studies are required to understand the pathogenesis of fungal infection in XLA.
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http://dx.doi.org/10.1007/s10875-021-01017-3DOI Listing
August 2021

Ensemble-based high-throughput virtual screening of natural ligands using the Super Natural-II database against cell-wall protein dTDP-4-dehydrorhamnose reductase (RmlD) in .

J Biomol Struct Dyn 2020 Dec 31:1-10. Epub 2020 Dec 31.

Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

The disease Tuberculosis (TB) is caused by a bacterium called (). The bacterial cell-wall consists of peptidoglycan layer maintains the cellular integrity and cell viability. The main problem resides in the cell cycle of in its quiescent form which is not targeted by any drugs hence there is an immediate need for new antibiotics to target the cell wall. The current study deals with the dTDP-4-dehydrorahmnose reductase (RmlD) which is the final enzyme in the series of cell-wall proteins of . The RmlD is a part of Carbohydrate biosynthesis has been considered as a good drug target for the novel class of antibiotics. Our study begins with the protein structure prediction, Homology studies were conducted using the Phyre2 web server. The structure is then refined and subjected to molecular dynamics simulations for 50 ns using GROMACS. The clustering analysis has been carried out and generated 41 clusters with 2 Å as the cut-off. Blind docking virtual screening was performed against RmlD protein using the Super Natural-II database with AutoDock4.0. its results helped to screen top ligands based on best binding energies. In both dockings, there are some common residues in which the ligands are interacting and forming the Hydrogen bonds such as Asp-105, Val-158, Thr-160, Gly-161, Arg-224, Arg-256. The ligand-567 giving the best results by being in the top-3 of all the clusters in both blind docking as well as the active-site docking. Hence ligand-567 can be a potential inhibitor of RmlD which can further inhibit the cell-wall synthesis of Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2020.1867641DOI Listing
December 2020

Whole exome sequencing identifies compound heterozygous variants of gene in monozygotic twin patients with common variable immunodeficiency.

SAGE Open Med 2020 22;8:2050312120922652. Epub 2020 May 22.

Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

Objectives: A pair of female Malay monozygotic twins who presented with recurrent upper respiratory tract infections, hepatosplenomegaly, bronchiectasis and bicytopenia were recruited in this study. Both patients were suspected with primary immunodeficiency diseases. However, the definite diagnosis was not clear due to complex disease phenotypes. The objective of this study was to identify the causative gene mutation in these patients.

Methods: Lymphocyte subset enumeration test and whole exome sequencing were performed.

Results: We identified a compound heterozygous mutation (c.1916G>A and c.2012G>A) in both patients. These variants were then confirmed using Sanger sequencing.

Conclusion: Whole exome sequencing analysis of the monozygotic twins revealed compound heterozygous missense mutations in .
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http://dx.doi.org/10.1177/2050312120922652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249565PMC
May 2020

A novel de novo NLRC4 mutation reinforces the likely pathogenicity of specific LRR domain mutation.

Clin Immunol 2020 02 20;211:108328. Epub 2019 Dec 20.

Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia; Centre of Research in Systems Biology, Structural Bioinformatics and Human Digital Imaging (CRYSTAL), University of Malaya, Kuala Lumpur, Malaysia. Electronic address:

Autoinflammatory disorders are characterized by dysregulated innate immune response, resulting in recurrent uncontrolled systemic inflammation and fever. Gain-of-function mutations in NLRC4 have been described to cause a range of autoinflammatory disorders. We report a twelve-year-old Malay girl with recurrent fever, skin erythema, and inflammatory arthritis. Whole exome sequencing and subsequent bidirectional Sanger sequencing identified a heterozygous missense mutation in NLRC4 (NM_001199138: c.1970A > T). This variant was predicted to be damaging in silico, was absent in public and local databases and occurred in a highly conserved residue in the leucine-rich repeat (LRR) domain. Cytokine analysis showed extremely high serum IL-18 and IL-18/CXCL9 ratio, consistent with other NLRC4-MAS patients. In summary, we identified the first patient with a novel de novo heterozygous NLRC4 gene mutation contributing to autoinflammatory disease in Malaysia. Our findings reinforce the likely pathogenicity of specific LRR domain mutations in NLRC4 and expand the clinical spectrum of NLRC4 mutations.
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http://dx.doi.org/10.1016/j.clim.2019.108328DOI Listing
February 2020

Transcriptogenomics identification and characterization of RNA editing sites in human primary monocytes using high-depth next generation sequencing data.

Genomics 2019 07 7;111(4):899-905. Epub 2018 Jun 7.

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; Centre of Research for Computational Sciences and Informatics in Biology, Bio11 Industry, Environment, Agriculture and Healthcare (CRYSTAL), University of Malaya, 50603 Kuala Lumpur, Malaysia.. Electronic address:

High-depth next generation sequencing data provide valuable insights into the number and distribution of RNA editing events. Here, we report the RNA editing events at cellular level of human primary monocyte using high-depth whole genomic and transcriptomic sequencing data. We identified over a ten thousand putative RNA editing sites and 69% of the sites were A-to-I editing sites. The sites enriched in repetitive sequences and intronic regions. High-depth sequencing datasets revealed that 90% of the canonical sites were edited at lower frequencies (<0.7). Single and multiple human monocytes and brain tissues samples were analyzed through genome sequence independent approach. The later approach was observed to identify more editing sites. Monocytes was observed to contain more C-to-U editing sites compared to brain tissues. Our results establish comparable pipeline that can address current limitations as well as demonstrate the potential for highly sensitive detection of RNA editing events in single cell type.
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http://dx.doi.org/10.1016/j.ygeno.2018.05.019DOI Listing
July 2019

Enzymatic inhibitory activity of leaf extract on matrix metalloproteinase-2, 8 and 9.

Nat Prod Res 2019 Jun 2;33(12):1765-1768. Epub 2018 Feb 2.

a Faculty of Science , Institute of Biological Sciences, University of Malaya , Kuala Lumpur , Malaysia.

Dysregulation of matrix metalloproteinases (MMPs) activity is known in many pathological conditions with which most of the conditions are related to elevate MMPs activities. (FD) is a plant known for its therapeutic properties. In order to evaluate the therapeutic potential of FD leaf extract, we study the enzymatic inhibition properties of FD leaf extract and its major bioactive compounds (vitexin and isovitexin) on a panel of MMPs (MMP-2, MMP-8 and MMP-9) using experimental and computational approaches. FD leaf extract and its major bioactive compounds showed pronounced inhibition activity towards the MMPs tested. Computational docking analysis revealed that vitexin and isovitexin bind to the active site of the three tested MMPs. We also evaluated the cytotoxicity and cell migration inhibition activity of FD leaf extract in the endothelial EA.hy 926 cell line. Conclusively, this study provided additional information on the potential of FD leaf extract for therapeutical application.
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http://dx.doi.org/10.1080/14786419.2018.1434631DOI Listing
June 2019

Transcriptome profiling of monocytes from XLA patients revealed the innate immune function dysregulation due to the BTK gene expression deficiency.

Sci Rep 2017 07 28;7(1):6836. Epub 2017 Jul 28.

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia.

X-linked agammaglobulinemia (XLA) is a rare genetic disorder, caused by mutations in BTK (Bruton's Tyrosine Kinase) gene. Deep high-throughput RNA sequencing (RNA-Seq) approach was utilized to explore the possible differences in transcriptome profiles of primary monocytes in XLA patients compared with healthy subjects. Our analysis revealed the differences in expression of 1,827 protein-coding genes, 95 annotated long non-coding RNAs (lncRNAs) and 20 novel lincRNAs between XLA patients and healthy subjects. GO and KEGG pathway analysis of differentially expressed (DE) protein-coding genes showed downregulation of several innate immune-related genes and upregulation of oxidative phosphorylation and apoptosis-related genes in XLA patients compared to the healthy subjects. Moreover, the functional prediction analysis of DE lncRNAs revealed their potential role in regulating the monocytes cell cycle and apoptosis in XLA patients. Our results suggested that BTK mutations may contribute to the dysregulation of innate immune system and increase susceptibility to apoptosis in monocytes of XLA patients. This study provides significant finding on the regulation of BTK gene in monocytes and the potential for development of innovative biomarkers and therapeutic monitoring strategies to increase the quality of life in XLA patients.
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http://dx.doi.org/10.1038/s41598-017-06342-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533715PMC
July 2017

Transcriptome landscape of human primary monocytes at different sequencing depth.

Genomics 2017 10 19;109(5-6):463-470. Epub 2017 Jul 19.

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; Centre of Research for Computational Sciences & Informatics for Biology, Bioindustry, Environment, Agriculture and Healthcare (CRYSTAL), University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address:

Differential gene and transcript expression pattern of human primary monocytes from healthy young subjects were profiled under different sequencing depths (50M, 100M, and 200M reads). The raw data consisted of 1.3 billion reads generated from RNA sequencing (RNA-Seq) experiments. A total of 17,657 genes and 75,392 transcripts were obtained at sequencing depth of 200M. Total splice junction reads showed an even more significant increase. Comparative analysis of the expression patterns of immune-related genes revealed a total of 217 differentially expressed (DE) protein-coding genes and 50 DE novel transcripts, in which 40 DE protein-coding genes were related to the immune system. At higher sequencing depth, more genes, known and novel transcripts were identified and larger proportion of reads were allowed to map across splice junctions. The results also showed that increase in sequencing depth has no effect on the sequence alignment.
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http://dx.doi.org/10.1016/j.ygeno.2017.07.003DOI Listing
October 2017

Experimental and computational approaches to reveal the potential of Ficus deltoidea leaves extract as α-amylase inhibitor.

Nat Prod Res 2018 Feb 10;32(4):473-476. Epub 2017 Apr 10.

a Bioinformatics Programme , Institute of Biological Sciences, Faculty of Science, University of Malaya , Kuala Lumpur , Malaysia.

Ficus deltoidea leaves extract are known to have good therapeutic properties such as antioxidant, anti-inflammatory and anti-diabetic. We showed that 50% ethanol-water extract of F. deltoidea leaves and its pungent compounds vitexin and isovitexin exhibited significant (p < 0.05) α-amylase inhibition with IC (vitexin: 4.6 μM [0.02 μg/mL]; isovitexin: 0.06 μg/mL [13.8 μM] and DPPH scavenging with IC (vitexin: 92.5 μM [0.4 μg/mL]; isovitexin: 0.5 μg/mL [115.4 μM]). Additionally, molecular docking analysis confirmed that vitexin has a higher binding affinity (-7.54 kcal/mol) towards α-amylase compared to isovitexin (-5.61 kcal/mol). On the other hand, the molecular dynamics findings showed that vitexin-α-amylase complex is more stable during the simulation of 20 ns when compared to the isovitexin-α-amylase complex. Our results suggest that vitexin is more potent and stable against α-amylase enzyme, thus it could develop as a therapeutic drug for the treatment of diabetes.
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http://dx.doi.org/10.1080/14786419.2017.1312393DOI Listing
February 2018

Toward a Reference Gene Catalog of Human Primary Monocytes.

OMICS 2016 11;20(11):627-634

1 Faculty of Science, Institute of Biological Sciences, University of Malaya , Kuala Lumpur, Malaysia .

Transcriptome analyses based on high-throughput RNA sequencing (RNA-Seq) provide powerful and quantitative characterization of cell types and in-depth understanding of biological systems in health and disease. In this study, we present a comprehensive transcriptome profile of human primary monocytes, a crucial component of the innate immune system. We performed deep RNA-Seq of monocytes from six healthy subjects and integrated our data with 10 other publicly available RNA-Seq datasets of human monocytes. A total of 1.9 billion reads were generated, which allowed us to capture most of the genes transcribed in human monocytes, including 11,994 protein-coding genes, 5558 noncoding genes (including long noncoding RNAs, precursor miRNAs, and others), 2819 pseudogenes, and 7034 putative novel transcripts. In addition, we profiled the expression pattern of 1155 transcription factors (TFs) in human monocytes, which are the main molecules in controlling the gene transcription. An interaction network was constructed among the top expressed TFs and their targeted genes, which revealed the potential key regulatory genes in biological function of human monocytes. The gene catalog of human primary monocytes provided in this study offers significant promise and future potential clinical applications in the fields of precision medicine, systems diagnostics, immunogenomics, and the development of innovative biomarkers and therapeutic monitoring strategies.
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http://dx.doi.org/10.1089/omi.2016.0124DOI Listing
November 2016

The conformational dynamics of H2-H3n and S2-H6 in gating ligand entry into the buried binding cavity of vitamin D receptor.

Sci Rep 2016 10 27;6:35937. Epub 2016 Oct 27.

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Crystal structures of holo vitamin D receptor (VDR) revealed a canonical conformation in which the ligand is entrapped in a hydrophobic cavity buried in the ligand-binding domain (LBD). The mousetrap model postulates that helix 12 is positioned away from the domain to expose the interior cavity. However, the extended form of helix 12 is likely due to artifacts during crystallization. In this study, we set out to investigate conformational dynamics of apo VDR using molecular dynamics simulation on microsecond timescale. Here we show the neighboring backbones of helix 2-helix 3n and beta strand 2-helix 6 of LBD, instead of the helix 12, undergo large-scale motion, possibly gating the entrance of ligand to the ligand binding domain. Docking analysis to the simulated open structure of VDR with the estimated free energy of -37.0 kJ/mol, would emphasise the role of H2-H3n and S2-H6 in facilitating the entrance of calcitriol to the LBD of VDR.
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http://dx.doi.org/10.1038/srep35937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081507PMC
October 2016

Long non-coding RNA expression in primary human monocytes.

Genomics 2016 07 8;108(1):37-45. Epub 2016 Jan 8.

Institute of Bioinformatics, International Technology Park, Bangalore, 560066, India; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address:

Long non-coding RNAs (lncRNAs) have been shown to possess a wide range of functions in both cellular and developmental processes including cancers. Although some of the lncRNAs have been implicated in the regulation of the immune response, the exact function of the large majority of lncRNAs still remains unknown. In this study, we characterized the lncRNAs in human primary monocytes, an essential component of the innate immune system. We performed RNA sequencing of monocytes from four individuals and combined our data with eleven other publicly available datasets. Our analysis led to identification of ~8000 lncRNAs of which >1000 have not been previously reported in monocytes. PCR-based validation of a subset of the identified novel long intergenic noncoding RNAs (lincRNAs) revealed distinct expression patterns. Our study provides a landscape of lncRNAs in monocytes, which could facilitate future experimental studies to characterize the functions of these molecules in the innate immune system.
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http://dx.doi.org/10.1016/j.ygeno.2016.01.002DOI Listing
July 2016

Amino acid sequence and structural comparison of BACE1 and BACE2 using evolutionary trace method.

ScientificWorldJournal 2014 28;2014:482463. Epub 2014 Aug 28.

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia ; Centre of Research for Computational Sciences and Informatics in Biology, Bioindustry, Environment, Agriculture and Healthcare (CRYSTAL), University of Malaya, 50603 Kuala Lumpur, Malaysia.

Beta-amyloid precursor protein cleavage enzyme 1 (BACE1) and beta-amyloid precursor protein cleavage enzyme 2 (BACE2), members of aspartyl protease family, are close homologues and have high similarity in their protein crystal structures. However, their enzymatic properties differ leading to disparate clinical consequences. In order to identify the residues that are responsible for such differences, we used evolutionary trace (ET) method to compare the amino acid conservation patterns of BACE1 and BACE2 in several mammalian species. We found that, in BACE1 and BACE2 structures, most of the ligand binding sites are conserved which indicate their enzymatic property of aspartyl protease family members. The other conserved residues are more or less randomly localized in other parts of the structures. Four group-specific residues were identified at the ligand binding site of BACE1 and BACE2. We postulated that these residues would be essential for selectivity of BACE1 and BACE2 biological functions and could be sites of interest for the design of selective inhibitors targeting either BACE1 or BACE2.
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http://dx.doi.org/10.1155/2014/482463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164807PMC
June 2015

A comparative analysis of synonymous codon usage bias pattern in human albumin superfamily.

ScientificWorldJournal 2014 20;2014:639682. Epub 2014 Feb 20.

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia ; Crystal, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Synonymous codon usage bias is an inevitable phenomenon in organismic taxa across the three domains of life. Though the frequency of codon usage is not equal across species and within genome in the same species, the phenomenon is non random and is tissue-specific. Several factors such as GC content, nucleotide distribution, protein hydropathy, protein secondary structure, and translational selection are reported to contribute to codon usage preference. The synonymous codon usage patterns can be helpful in revealing the expression pattern of genes as well as the evolutionary relationship between the sequences. In this study, synonymous codon usage bias patterns were determined for the evolutionarily close proteins of albumin superfamily, namely, albumin, α-fetoprotein, afamin, and vitamin D-binding protein. Our study demonstrated that the genes of the four albumin superfamily members have low GC content and high values of effective number of codons (ENC) suggesting high expressivity of these genes and less bias in codon usage preferences. This study also provided evidence that the albumin superfamily members are not subjected to mutational selection pressure.
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http://dx.doi.org/10.1155/2014/639682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951064PMC
January 2015

A novel Bruton's tyrosine kinase gene (BTK) invariant splice site mutation in a Malaysian family with X-linked agammaglobulinemia.

Asian Pac J Allergy Immunol 2013 Dec;31(4):320-4

Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

X-linked agammaglobulinemia (XLA) is a rare genetic disorder caused by mutations in the Bruton's tyrosine kinase (BTK) gene. These mutations cause defects in early B cell development. A patient with no circulating B cells and low serum immunoglobulin isotypes was studied as were his mother and sister. Monocyte BTK protein expression was evaluated by flow cytometry. The mutation was determined using PCR and followed by sequencing. Flow cytometry showed the patient lacked BTK protein expression in his monocytes while the mother and sister had 62% and 40% of the monocytes showing BTK protein expressions respectively. The patient had a novel base substitution in the first nucleotide of intron 9 in the BTK gene, and the mutation was IVS9+1G
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http://dx.doi.org/10.12932/AP0304.31.4.2013DOI Listing
December 2013

A combination of evolutionary trace method, molecular surface accessibility and hydrophobicity analysis to design a high hydrophobicity laccase.

In Silico Biol 2010 ;10(3):145-53

Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia.

Laccases are industrially attractive enzymes and their applications have expanded to the field of bioremediation. The challenge of today's biotechnology in enzymatic studies is to design enzymes that not only have a higher activity but are also more stable and could fit well with the condition requirements. Laccases are known to oxidize non-natural substrates like polycyclic aromatic hydrocarbons (PAHs). We suppose by increasing the hydrophobicity of laccase, it would increase the chance of the enzyme to meet the hydrophobic substrates in a contamination site, therefore increasing the bioremediation efficacy of PAHs from environment. In this attempt, the applications of evolutionary trace (ET), molecular surface accessibility and hydrophobicity analysis on laccase sequences and laccase's crystal structure (1KYA) are described for optimal design of an enzyme with higher hydrophobicity. Our analysis revealed that Q23A, Q45I, N141A, Q237V, N262L, N301V, N331A, Q360L and Q482A could be promising exchanges to be tested in mutagenesis experiments.
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http://dx.doi.org/10.3233/ISB-2010-0423DOI Listing
September 2013

Evolutionary trace analysis at the ligand binding site of laccase.

Bioinformation 2008 Jun 18;2(9):369-72. Epub 2008 Jun 18.

Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Laccase belongs to the family of blue multi-copper oxidases and are capable of oxidizing a wide range of aromatic compounds. Laccases have industrial applications in paper pulping or bleaching and hydrocarbon bioremediation as a biocatalyst. We describe the design of a laccase with broader substrate spectrum in bioremediation. The application of evolutionary trace (ET) analysis of laccase at the ligand binding site for optimal design of the enzyme is described. In this attempt, class specific sites from ET analysis were mapped onto known crystal structure of laccase. The analysis revealed 162PHE as a critical residue in structure function relationship studies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2533054PMC
http://dx.doi.org/10.6026/97320630002369DOI Listing
June 2008

Gc protein-derived macrophage activating factor (GcMAF): isoelectric focusing pattern and tumoricidal activity.

Anticancer Res 2003 Nov-Dec;23(6a):4451-7

Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Minamijyosajimacho-2, Tokushima, 770-8506 Japan.

Background: Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition.

Materials And Methods: We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release.

Results: The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected.

Conclusion: The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.
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January 2004

Preparation of Gc protein-derived macrophage activating factor (GcMAF) and its structural characterization and biological activities.

Anticancer Res 2002 Nov-Dec;22(6C):4297-300

Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Minamijyosanjimacho-2, Tokushima, 770-8506 Japan.

Background: Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF.

Materials And Methods: Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay.

Results: We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity.

Conclusion: Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.
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March 2003
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