Hydrophilic residues surrounding the S1 and S2 pockets contribute to dimerisation and catalysis in human dipeptidyl peptidase 8 (DP8)

2010 ◽  
Vol 391 (8) ◽  
Author(s):  
Melissa R. Pitman ◽  
R. Ian Menz ◽  
Catherine A. Abbott
Keyword(s):  
Biological Function ◽  
Homology Modelling ◽  
Dipeptidyl Peptidase ◽  
Fibroblast Activation Protein ◽  
Size Exclusion ◽  
Dimer Interface ◽  
Endopeptidase Activity ◽  
Exclusion Chromatography ◽  
Hydrophilic Residues ◽  
Substrate Kinetics

Abstract Dipeptidyl peptidase (DP) 8 belongs to the dipeptidyl peptidase IV gene family. DP8 has been implicated in immune function and asthma, although its biological function is yet unknown. Structures of the homologs, fibroblast activation protein (FAP) and DPIV, are known but the DP8 structure is yet to be resolved. To help characterise the DP8 substrate pocket, mutants of residues lining the pocket were produced at DP8D772, DP8Y315, DP8H434 and DP8D435 and assessed by substrate kinetics and size-exclusion chromatography. Mutations of DP8D772A/E/S/V affected catalysis but did not confer endopeptidase activity. Mutations of DP8H434F, DP8D435F and DP8Y315F reduced catalytic activity. Furthermore, mutations to DP8D772A/E/S/V, DP8H434F, DP8D435F and DP8Y315F affected dimer stabilisation. Homology modelling of DP8 using DPIV and FAP crystal structures suggested that DP8D772, DP8H434 and DP8D435 were located at the edge of the S2 catalytic pocket, contributing to the junction between the alpha-beta hydrolase and beta-propeller domains. This study provides insights into how the DP8 substrate pocket and dimer interface differ from DPIV and FAP which could be utilised for designing more selective DP8 inhibitors.

Purification and characterization of guanylate kinase, a nucleoside monophosphate kinase of Brugia malayi

Parasitology ◽  
2014 ◽  
Vol 141 (10) ◽  
pp. 1341-1352 ◽  
Author(s):  
SMITA GUPTA ◽  
SUNITA YADAV ◽  
NIDHI SINGH ◽  
ANITA VERMA ◽  
IMRAN SIDDIQI ◽  
...  
Keyword(s):  
Homology Modelling ◽  
Substrate Binding ◽  
Brugia Malayi ◽  
Catalytic Efficiency ◽  
Size Exclusion ◽  
Kinetic Properties ◽  
Guanylate Kinase ◽  
Native Molecular Mass ◽  
Exclusion Chromatography ◽  
Kinase A

SUMMARYGuanylate kinase, a nucleoside monophosphate kinase of Brugia malayi which is involved in reversible transfer of phosphate groups from ATP to GMP, was cloned, expressed and characterized. The native molecular mass of BmGK was found to be 45 kDa as determined by size exclusion chromatography and glutaraldehyde cross-linking which revealed that the protein is homodimer in nature. This is a unique characteristic among known eukaryotic GKs. GMP and ATP served as the most effective phosphate acceptor and donor, respectively. Recombinant BmGK utilized both GMP and dGMP, as substrates showing Km values of 30 and 38 μm, respectively. Free Mg+2 (un-complexed to ATP) and GTP play a regulatory role in catalysis of BmGK. The enzyme showed higher catalytic efficiency as compared with human GK and showed ternary complex (BmGK-GMP-ATP) formation with sequential substrate binding. The secondary structure of BmGK consisted of 45% α-helices, 18% β-sheets as revealed by CD analysis. Homology modelling and docking with GMP revealed conserved substrate binding residues with slight differences. Differences in kinetic properties and oligomerization of BmGK compared with human GK can provide the way for design of parasite-specific inhibitors.

scholarly journals High-grade extracellular vesicles preparation by combined size-exclusion and affinity chromatography

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Cristina Bellotti ◽  
Kristina Lang ◽  
Nataliya Kuplennik ◽  
Alejandro Sosnik ◽  
Robert Steinfeld
Keyword(s):  
Affinity Chromatography ◽  
Extracellular Vesicles ◽  
Biological Function ◽  
Therapeutic Potential ◽  
Folate Receptor ◽  
Size Exclusion ◽  
Selective Enrichment ◽  
Large Numbers ◽  
Marker Proteins ◽  
Exclusion Chromatography

AbstractExtracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of EVs such as exosomes and ectosomes, which are involved in distinct biological processes. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of human dipeptidyl peptidase 10 (DPPY), a component of voltage-gated potassium channels

2012 ◽  
Vol 68 (2) ◽  
pp. 214-217 ◽  
Author(s):  
Gustavo Arruda Bezerra ◽  
Elena Dobrovetsky ◽  
Alma Seitova ◽  
Sirano Dhe-Paganon ◽  
Karl Gruber
Keyword(s):  
Potassium Channels ◽  
Surface Expression ◽  
Dipeptidyl Peptidase ◽  
Cell System ◽  
Size Exclusion ◽  
Cell Surface Expression ◽  
Electrophysiological Properties ◽  
Cell Parameters ◽  
Voltage Gated ◽  
Exclusion Chromatography

Dipeptidyl peptidase 10 (DPP10, DPPY) is an inactive peptidase associated with voltage-gated potassium channels, acting as a modulator of their electrophysiological properties, cell-surface expression and subcellular localization. Because potassium channels are important disease targets, biochemical and structural characterization of their interaction partners was sought. DPP10 was cloned and expressed using an insect-cell system and the protein was purifiedvia His-tag affinity and size-exclusion chromatography. Crystals obtained by the sitting-drop method were orthorhombic, belonging to space groupP212121with unit-cell parametersa= 80.91,b= 143.73,c= 176.25 Å. A single solution with two molecules in the asymmetric unit was found using the structure of DPP6 (also called DPPX; PDB entry 1xfd) as the search model in a molecular replacement protocol.

scholarly journals High-grade Extracellular Vesicles Preparation by Combined Size- exclusion and Affinity Chromatography

2021 ◽  
Author(s):  
Cristina Bellotti ◽  
Kristina Lang ◽  
Nataliya Kuplennik ◽  
Alejandro Sosnik ◽  
Robert Steinfeld
Keyword(s):  
Affinity Chromatography ◽  
Extracellular Vesicles ◽  
Biological Function ◽  
Therapeutic Potential ◽  
Folate Receptor ◽  
Size Exclusion ◽  
Selective Enrichment ◽  
Large Numbers ◽  
Marker Proteins ◽  
Exclusion Chromatography

Abstract Extracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of EVs such as exosomes, microvesicles, microparticles, and ectosomes which are involved in distinct biological processes. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

Structure of a shear-thickening polysaccharide extracted from the New Zealand black tree fern, Cyathea medullaris

2020 ◽  
Author(s):  
M Wee ◽  
M Mastrangelo ◽  
Susan Carnachan ◽  
Ian Sims ◽  
K Goh
Keyword(s):  
New Zealand ◽  
Uronic Acid ◽  
Average Molecular Weight ◽  
Shear Thickening ◽  
Water Soluble ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Tree Fern ◽  
13C Nuclear Magnetic Resonance ◽  
Exclusion Chromatography

A shear-thickening water-soluble polysaccharide was purified from mucilage extracted from the fronds of the New Zealand black tree fern (Cyathea medullaris or 'mamaku' in Māori) and its structure characterised. Constituent sugar analysis by three complementary methods, combined with linkage analysis (of carboxyl reduced samples) and 1H and 13C nuclear magnetic resonance spectroscopy (NMR) revealed a glucuronomannan comprising a backbone of 4-linked methylesterified glucopyranosyl uronic acid and 2-linked mannopyranosyl residues, branched at O-3 of 45% and at both O-3 and O-4 of 53% of the mannopyranosyl residues with side chains likely comprising terminal xylopyranosyl, terminal galactopyranosyl, non-methylesterified terminal glucopyranosyl uronic acid and 3-linked glucopyranosyl uronic acid residues. The weight-average molecular weight of the purified polysaccharide was ~1.9×106Da as determined by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS). The distinctive rheological properties of this polysaccharide are discussed in relation to its structure. © 2014 Elsevier B.V.

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