Tailoring GalNAcα1-3Galβ-specific lectins from a multi-specific fungal galectin: dramatic change of carbohydrate specificity by a single amino-acid substitution

2013 ◽  
Vol 453 (2) ◽  
pp. 261-270 ◽  
Author(s):  
Dan Hu ◽  
Hiroaki Tateno ◽  
Takashi Sato ◽  
Hisashi Narimatsu ◽  
Jun Hirabayashi
Keyword(s):  
Dissociation Constants ◽  
Multiple Roles ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Dramatic Improvement ◽  
Carbohydrate Specificity ◽  
Cell Staining ◽  
Group A

Galectins exhibit multiple roles through recognition of diverse structures of β-galactosides. However, this broad specificity often hinders their practical use as probes. In the present study we report a dramatic improvement in the carbohydrate specificity of a multi-specific fungal galectin from the mushroom Agrocybe cylindricea, which binds not only to simple β-galactosides, but also to their derivatives. Site-directed mutagenesis targeting five residues involved in β-galactose binding revealed that replacement of Asn46 with alanine (N46A) increased the binding to GalNAcα1-3Galβ-containing glycans, while eliminating binding to all other β-galactosides, as shown by glycoconjugate microarray analysis. Quantitative analysis by frontal affinity chromatography showed that the mutant N46A had enhanced affinity towards blood group A tetraose (type 2), A hexaose (type 1) and Forssman pentasaccharide with dissociation constants of 5.0×10−6 M, 3.8×10−6 M and 1.0×10−5 M respectively. Surprisingly, all the other mutants generated by saturation mutagenesis of Asn46 exhibited essentially the same specificity as N46A. Moreover, alanine substitution for Pro45, which forms the cis-conformation upon β-galactose binding, exhibited the same specificity as N46A. From a practical viewpoint, the derived N46A mutant proved to be unique as a specific probe to detect GalNAcα1-3Galβ-containing glycans by methods such as flow cytometry, cell staining and lectin microarray.

scholarly journals A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

2021 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth
Keyword(s):  
Single Point ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Single Amino Acid Residue

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

scholarly journals Characterization of FimH Adhesins Expressed by Salmonella enterica Serovar Gallinarum Biovars Gallinarum and Pullorum: Reconstitution of Mannose-Binding Properties by Single Amino Acid Substitution

2005 ◽  
Vol 73 (9) ◽  
pp. 6187-6190 ◽  
Author(s):  
Dagmara Kisiela ◽  
Anna Sapeta ◽  
Maciej Kuczkowski ◽  
Tadeusz Stefaniak ◽  
Alina Wieliczko ◽  
...  
Keyword(s):  
Colon Carcinoma ◽  
Salmonella Enterica ◽  
Human Colon ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Binding Properties ◽  
Type 1 Fimbriae ◽  
Serovar Typhimurium ◽  
High Mannose

ABSTRACT Recombinant FimH adhesins of type 1 fimbriae from Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, in contrast to those of Salmonella enterica serovar Typhimurium, did not bind to high-mannose oligosaccharides or to human colon carcinoma HT-29 cells. However, mutated FimH proteins from biovar Gallinarum and biovar Pullorum, in which the isoleucine at position 78 was replaced by the threonine found in S. enterica serovar Typhimurium, bound well to glycoproteins carrying high-mannose oligosaccharides and colon carcinoma cells. The loss of sugar-binding properties by biovar Gallinarum and biovar Pullorum FimH adhesins, which are a part of the type 1 fimbriae, is most probably the result of a single T78I mutation, as was proven by site-directed mutagenesis of FimH proteins.

Comparative Study of effectiveness of mobilization with movement (MWM) and End range mobilization (ERM) techniques in frozen shoulder

2018 ◽  
Vol 4 (4) ◽  
pp. 519-522
Author(s):  
Jeyakumar S ◽  
Jagatheesan Alagesan ◽  
T.S. Muthukumar
Keyword(s):  
Range Of Motion ◽  
Frozen Shoulder ◽  
Type I ◽  
Mean Values ◽  
Functional Activities ◽  
Group A ◽  
The Mean ◽  
Group B

Background: Frozen shoulder is disorder of the connective tissue that limits the normal Range of motion of the shoulder in diabetes, frozen shoulder is thought to be caused by changes to the collagen in the shoulder joint as a result of long term Hypoglycemia. Mobilization is a therapeutic movement of the joint. The goal is to restore normal joint motion and rhythm. The use of mobilization with movement for peripheral joints was developed by mulligan. This technique combines a sustained application of manual technique “gliding” force to the joint with concurrent physiologic motion of joint, either actively or passively. This study aims to find out the effects of mobilization with movement and end range mobilization in frozen shoulder in Type I diabetics. Materials and Methods: 30 subjects both male and female, suffering with shoulder pain and clinically diagnosed with frozen shoulder was recruited for the study and divided into two groups with 15 patients each based on convenient sampling method. Group A patients received mobilization with movement and Group B patients received end range mobilization for three weeks. The outcome measurements were SPADI, Functional hand to back scale, abduction range of motion using goniometer and VAS. Results: The mean values of all parameters showed significant differences in group A as compared to group B in terms of decreased pain, increased abduction range and other outcome measures. Conclusion: Based on the results it has been concluded that treating the type 1 diabetic patient with frozen shoulder, mobilization with movement exercise shows better results than end range mobilization in reducing pain and increase functional activities and mobility in frozen shoulder.

scholarly journals Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

2009 ◽  
Vol 90 (7) ◽  
pp. 1741-1747 ◽  
Author(s):  
Tahir H. Malik ◽  
Candie Wolbert ◽  
Laura Nerret ◽  
Christian Sauder ◽  
Steven Rubin
Keyword(s):  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Change ◽  
Mumps Virus ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Supporting Evidence ◽  
L Protein ◽  
Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

scholarly journals Mutation of Dileucine-Like Motifs in the Human Immunodeficiency Virus Type 1 Capsid Disrupts Virus Assembly, Gag-Gag Interactions, Gag-Membrane Binding, and Virion Maturation

2006 ◽  
Vol 80 (16) ◽  
pp. 7939-7951 ◽  
Author(s):  
Anjali Joshi ◽  
Kunio Nagashima ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Virus Assembly ◽  
Membrane Binding ◽  
Single Amino Acid ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein Pr55Gag drives the assembly and release of virus-like particles in the infected cell. The capsid (CA) domain of Gag plays an important role in these processes by promoting Gag-Gag interactions during assembly. The C-terminal domain (CTD) of CA contains two dileucine-like motifs (L189/L190 and I201/L202) implicated in regulating the localization of Gag to multivesicular bodies (MVBs). These dileucine-like motifs are located in the vicinity of the CTD dimer interface, a region of CA critical for Gag-Gag interactions during virus assembly and CA-CA interactions during core formation. To study the importance of the CA dileucine-like motifs in various aspects of HIV-1 replication, we introduced a series of mutations into these motifs in the context of a full-length, infectious HIV-1 molecular clone. CA mutants LL189,190AA and IL201,202AA were both severely impaired in virus particle production because of a variety of defects in the binding of Gag to membrane, Gag multimerization, and CA folding. In contrast to the model suggesting that the CA dileucine-like motifs regulate MVB targeting, the IL201,202AA mutation did not alter Gag localization to the MVB in either HeLa cells or macrophages. Revertants of single-amino-acid substitution mutants were obtained that no longer contained dileucine-like motifs but were nevertheless fully replication competent. The varied phenotypes of the mutants reported here provide novel insights into the interplay among Gag multimerization, membrane binding, virus assembly, CA dimerization, particle maturation, and virion infectivity.

Identification of Type 2 von Willebrand Disease in Previously Diagnosed Type 1 Patients: a Reappraisal Using Phenotypes, Genotypes and Molecular Modelling

2000 ◽  
Vol 84 (12) ◽  
pp. 998-1004 ◽  
Author(s):  
Ioana Nitu-Whalley ◽  
Anne Riddell ◽  
K. Pasi ◽  
Dale Owens ◽  
M. Enayat ◽  
...  
Keyword(s):  
Molecular Modelling ◽  
Correct Diagnosis ◽  
Von Willebrand Disease ◽  
Single Amino Acid ◽  
Elisa Assay ◽  
Modelling Analysis ◽  
A1 Domain ◽  
Von Willebrand

SummaryIn order to investigate the possibility that qualitative type 2 defects in von Willebrand factor (VWF) occurred in patients previously diagnosed with quantitative type 1 von Willebrand disease (VWD), the phenotypes and genotypes were reanalysed in 30 patients who exhibited discrepant VWF activity/VWF:Ag ratios of less than 0.7. The capacity of VWF to bind to glycoprotein Ib (GpIb) was reassessed using the ristocetin co-factor activity (VWF:RiCo) assay compared to an in-house and a commercial ELISA assay (based on a mAb directed against the GpIb binding site on VWF). This was supplemented by multimeric analysis and the amplification and sequencing of a 936 bp fragment of exon 28 of the VWF gene with the aim of identifying mutations in the A1 domain. On reappraisal, using the VWF:RiCo assay all patients demonstrated a disproportionately reduced VWF:RiCo/VWF:Ag ratio, indicative of a qualitative defect, while abnormal ratios were detected in only seven kindreds using the in-house ELISA assay and in only one kindred with the commercial ELISA assay. Eight single amino acid substitutions were found in nine kindreds, four of which were novel candidate VWF mutations and four previously described in association with type 2 VWD. In agreement with the phenotype, the novel VWF mutations were located in the VWF-A1 crystal structure at positions that corresponded to potential type 2M defects. This study underlines the difficulties of correct diagnosis of the subtype of VWD and emphasises the importance of using sensitive phenotypic assays, the relevance of the VWF:RiCo/ VWF:Ag ratio, multimeric analysis and molecular modelling analysis.

scholarly journals The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

2017 ◽  
Vol 399 (1) ◽  
pp. 73-77 ◽  
Author(s):  
Monika B. Dolinska ◽  
Yuri V. Sergeev
Keyword(s):  
Oculocutaneous Albinism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Insect Larvae ◽  
Melanin Biosynthesis ◽  
Human Tyrosinase

AbstractTyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studiedin vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residuesin vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

scholarly journals Comparison of Predicted Scaffold-Compatible Sequence Variation in the Triple-Hairpin Structure of Human Immunodeficiency Virus Type 1 gp41 with Patient Data

2002 ◽  
Vol 76 (15) ◽  
pp. 7595-7606 ◽  
Author(s):  
Nathalie Boutonnet ◽  
Wouter Janssens ◽  
Carlo Boutton ◽  
Jean-Luc Verschelde ◽  
Leo Heyndrickx ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Sequence Variation ◽  
Single Point ◽  
Point Mutations ◽  
Hairpin Structure ◽  
Single Amino Acid ◽  
Immunodeficiency Virus

ABSTRACT It has been proposed that the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 (e-gp41), involved in HIV entry into the target cell, exists in at least two conformations, a pre-hairpin intermediate and a fusion-active hairpin structure. To obtain more information on the structure-sequence relationship in e-gp41, we performed in silico a full single-amino-acid substitution analysis, resulting in a Fold Compatible Database (FCD) for each conformation. The FCD contains for each residue position in a given protein a list of values assessing the energetic compatibility (ECO) of each of the 20 natural amino acids at that position. Our results suggest that FCD predictions are in good agreement with the sequence variation observed for well-validated e-gp41 sequences. The data show that at a minECO threshold value of 5 kcal/mol, about 90% of the observed patient sequence variation is encompassed by the FCD predictions. Some inconsistent FCD predictions at N-helix positions packing against residues of the C helix suggest that packing of both peptides may involve some flexibility and may be attributed to an altered orientation of the C-helical domain versus the N-helical region. The permissiveness of sequence variation in the C helices is in agreement with FCD predictions. Comparison of N-core and triple-hairpin FCDs suggests that the N helices may impose more constraints on sequence variation than the C helices. Although the observed sequences of e-gp41 contain many multiple mutations, our method, which is based on single-point mutations, can predict the natural sequence variability of e-gp41 very well.

scholarly journals Immunochemical studies on blood groups LXII. Fractionation of hog and human A, H, and AH blood group active substance on insoluble immunoadsorbents of Dolichos and Lotus lectins.

1976 ◽  
Vol 143 (2) ◽  
pp. 422-436 ◽  
Author(s):  
M E Pereira ◽  
E A Kabat
Keyword(s):  
Blood Group ◽  
Optical Rotation ◽  
Gastric Mucin ◽  
Specific Optical Rotation ◽  
Dolichos Biflorus ◽  
Blood Group A ◽  
Analytical Properties ◽  
Group Substance ◽  
Group A

The purified lectins from Lotus tetragonolobus and Dolichos biflorus were coupled to Sepharose 2B to make insoluble adsorbents for purification and fractionation of blood group A and H active glycoproteins. With both adsorbents, hog gastric mucin A + H blood substance (HGM), purified by phenol-ethanol precipitation, yielded fractions showing only A, only H, or AH activities. The AH fraction was obtained when the adsorbent column was overloaded with HGM and its A and H specificities seem to be carried on the same molecules since they were not separable by chromatography on either column. However A and H specificities of blood group substance from the stomach of a presumably heterozygous individual hog were both on the same molecules as they too could not be fractionated on either column. Analytical properties of the isolated fractions were generally similar to those of the unfractionated material, the purfied A substances had a higher galactosamine/fucose ratio than did the H substances. Although the original A + H showed very little specific optical rotation, the separated A and H substances rotated positively and negatively, respectively. The lectin-Sepharose adsorbents have also proven useful in isolating A or H substances directly from the crude commercial hog gastric mucin. Blood group A2 substance from a human ovarian cyst yielded two fractions on the Lotus-Sepharose column; the effluent did not interact with the Lotus lectin but precipitated the Ulex and Dolichos lectins and anti-A, and appears to contain type 1 H determinants. The other fraction reacted with Lotus and Ulex lectin as well as with Dolichos and anti-A.

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