scholarly journals Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain

2003 ◽  
Vol 373 (2) ◽  
pp. 369-379 ◽  
Author(s):  
Maria-Dolores MONTIEL ◽  
Marie-Ange KRZEWINSKI-RECCHI ◽  
Philippe DELANNOY ◽  
Anne HARDUIN-LEPERS
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Human Gene ◽  
Short Form ◽  
Cytoplasmic Tail ◽  
Gene Organization ◽  
Sialic Acid Residue ◽  
Chromosome 17 ◽  
Soluble Form ◽  
Amino Acid Residues

The human Sda antigen is formed through the addition of an N-acetylgalactosamine residue via a β1,4-linkage to a sub-terminal galactose residue substituted with an α2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acα2-3Galβ-R β1,4-N-acetylgalactosaminyltransferase (Sda β1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sda β1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named E1S and E1L, leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74% identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to α2,3-sialylated acceptor substrates, to form the GalNAcβ1-4[Neu5Acα2-3]Galβ1-R trisaccharide common to both Sda and Cad antigens.

scholarly journals A Novel Cytoplasmic Tail MXXXL Motif Mediates the Internalization of Prostate-specific Membrane Antigen

2003 ◽  
Vol 14 (12) ◽  
pp. 4835-4845 ◽  
Author(s):  
Sigrid A. Rajasekaran ◽  
Gopalakrishnapillai Anilkumar ◽  
Eri Oshima ◽  
James U. Bowie ◽  
He Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Protein ◽  
Interleukin 2 ◽  
Adaptor Protein ◽  
Cytoplasmic Tail ◽  
Prostate Specific Membrane Antigen ◽  
Amino Acid Residues ◽  
Terminal Amino ◽  
Specific Membrane

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the α-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative μ2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.

scholarly journals Bioinformatics Insights Into Microbial Xylanase Protein Sequences

2018 ◽  
Vol 15 (2) ◽  
pp. 275-294
Author(s):  
Deepsikha Anand ◽  
Jeya Nasim ◽  
Sangeeta Yadav ◽  
Dinesh Yadav
Keyword(s):  
Amino Acid ◽  
Phylogenetic Tree ◽  
Catalytic Domain ◽  
Genetic Manipulation ◽  
Protein Sequences ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Molecular Weights ◽  
Aliphatic Index ◽  
Chemical Attributes

Microbial xylanases represents an industrially important group of enzymes associated with hydrolysis of xylan, a major hemicellulosic component of plant cell walls. A total of 122 protein sequences comprising of 58 fungal, 25 bacterial, 19actinomycetes and 20 yeasts xylanaseswere retrieved from NCBI, GenBank databases. These sequences were in-silico characterized for homology,sequence alignment, phylogenetic tree construction, motif assessment and physio-chemical attributes. The amino acid residues ranged from 188 to 362, molecular weights were in the range of 20.3 to 39.7 kDa and pI ranged from 3.93 to 9.69. The aliphatic index revealed comparatively less thermostability and negative GRAVY indicated that xylanasesarehydrophilicirrespective of the source organisms.Several conserved amino acid residues associated with catalytic domain of the enzyme were observed while different microbial sources also revealed few conserved amino acid residues. The comprehensive phylogenetic tree indicatedsevenorganismsspecific,distinct major clusters,designated as A, B, C, D, E, F and G. The MEME based analysis of 10 motifs indicated predominance of motifs specific to GH11 family and one of the motif designated as motif 3 with sequence GTVTSDGGTYDIYTTTRTNAP was found to be present in most of the xylanases irrespective of the sources.Sequence analysis of microbial xylanases provides an opportunity to develop strategies for molecular cloning and expression of xylanase genes and also foridentifying sites for genetic manipulation for developing novel xylanases with desired features as per industrial needs.

scholarly journals The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail

1993 ◽  
Vol 106 (3) ◽  
pp. 831-846 ◽  
Author(s):  
J. Pieters ◽  
O. Bakke ◽  
B. Dobberstein
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Mhc Class Ii ◽  
Cytoplasmic Tail ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Amino Acid Residues ◽  
Marker Proteins ◽  
Membrane Spanning

The oligomeric complex formed by major histocompatibility complex (MHC) class II alpha and beta chains and invariant chain (Ii) assembles in the endoplasmic reticulum and is then transported via the Golgi complex to compartments of the endocytic pathway. When Ii alone is expressed in CV1 cells it is sorted to endosomes. The Ii cytoplasmic tail has been found to be essential for targeting to these compartments. In order to characterize further the signals responsible for endosomal targeting, we have deleted various segments of the cytoplasmic tail. The Ii mutants were transiently expressed and the cellular location of the proteins was analyzed biochemically and morphologically. The cytoplasmic tail of Ii was found to contain two endosomal targeting sequences within its cytoplasmic tail; one targeting sequence was present within amino acid residues 12–29 and deletion of this segment revealed the presence of a second endosomal targeting sequence, located within the first 11 amino acid residues. The presence of a leucine-isoleucine pair at positions 7 and 8 within this sequence was found to be essential for endosomal targeting. In addition, the presence of this L-I motif lead to accumulation of Ii molecules in large endosomal vacuoles containing lysosomal marker proteins. Both wild type Ii and Ii mutant molecules containing only one endosomal targeting sequence were rapidly internalized from the plasma membrane. When the Ii cytoplasmic tail was fused to the membrane-spanning region of neuraminidase, a resident plasma membrane protein, the resulting chimera (INA) was found in endocytic compartments containing lysosomal marker proteins. Thus the cytoplasmic tail of Ii is sufficient for targeting to the endocytic/lysosomal pathway.

scholarly journals Cloning and Sequence Analysis of a New Cellulase Gene Encoding CelK, a Major Cellulosome Component of Clostridium thermocellum: Evidence for Gene Duplication and Recombination

1999 ◽  
Vol 181 (17) ◽  
pp. 5288-5295 ◽  
Author(s):  
Irina Kataeva ◽  
Xin-Liang Li ◽  
Huizhong Chen ◽  
Sang-Ki Choi ◽  
Lars G. Ljungdahl
Keyword(s):  
Amino Acid ◽  
Gene Duplication ◽  
Signal Peptide ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Size Difference ◽  
Amino Acid Residues ◽  
Cellulase Gene ◽  
Original Activity

ABSTRACT The cellulolytic and hemicellulolytic complex of Clostridium thermocellum, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide. We report here a new cellulase gene, celK, coding for CelK, a 98-kDa major component of the cellulosome. The gene has an open reading frame (ORF) of 2,685 nucleotides coding for a polypeptide of 895 amino acid residues with a calculated mass of 100,552 Da. A signal peptide of 27 amino acid residues is cut off during secretion, resulting in a mature enzyme of 97,572 Da. The nucleotide sequence is highly similar to that of cbhA(V. V. Zverlov et al., J. Bacteriol. 180:3091–3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium. Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid levels, respectively. Both have domain structures consisting of a signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain. A striking distinction between the two polypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-like domain and family III CBD. This insertion, missing in CelK, is responsible for the size difference between CelK and CbhA. Upstream and downstream flanking sequences of the two genes show no homology. The data indicate thatcelK and cbhA in the genome of C. thermocellum have evolved through gene duplication and recombination of domain coding sequences. celK without a dockerin domain was expressed in Escherichia coli and purified. The enzyme had pH and temperature optima at 6.0 and 65°C, respectively. It hydrolyzedp-nitrophenyl-β-d-cellobioside with aKm and a V max of 1.67 μM and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of CelK activity, with a Ki of 0.29 mM. The enzyme was thermostable, after 200 h of incubation at 60°C, 97% of the original activity remained. Properties of the enzyme indicated that it is a cellobiohydrolase.

scholarly journals Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

2002 ◽  
Vol 184 (9) ◽  
pp. 2399-2403 ◽  
Author(s):  
Fumiyoshi Okazaki ◽  
Yutaka Tamaru ◽  
Shinnosuke Hashikawa ◽  
Yu-Teh Li ◽  
Toshiyoshi Araki
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Catalytic Domain ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Binding Studies ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A β-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the Kd and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan.

scholarly journals Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil.

2016 ◽  
Vol 63 (1) ◽  
Author(s):  
Monika Wicka ◽  
Marta Wanarska ◽  
Ewelina Krajewska ◽  
Anna Pawlak-Szukalska ◽  
Józef Kur ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Pseudomonas Sp ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Optimal Activity ◽  
Cold Active ◽  
Psychrotolerant Bacterium ◽  
Type V

An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.

Cloning and sequencing of two genes encoding chitinases A and B from Bacillus cereus CH

2001 ◽  
Vol 47 (10) ◽  
pp. 895-902 ◽  
Author(s):  
Naoto Mabuchi ◽  
Yoshio Araki
Keyword(s):  
Amino Acid ◽  
Bacillus Cereus ◽  
Catalytic Domain ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Amino Acid Residues ◽  
Chitinase A ◽  
Genes Encoding ◽  
Fibronectin Type Iii ◽  
Polypeptide Chains

Two genes encoding chitinases A and B (chiA and chiB) from Bacillus cereus CH were cloned into Escherichia coli XL1-Blue MRF' by using pBluescript II SK+, and their nucleotide sequences were determined. Open reading frames of the chiA and chiB genes encoded distinct polypeptide chains consisting of 360 and 674 amino acid residues, respectively, with calculated molecular sizes of 39 470 and 74 261 Da, respectively. Comparison of the deduced amino acid sequences with those of other bacterial chitinases revealed that chitinase A consisted of a catalytic domain, while chitinase B consisted of three functional domains, a catalytic domain, a fibronectin type III-like domain, and a cellulose-binding domain. The primary structures of these two proteins were not similar to each other.Key words: Bacillus cereus, chitinase, cloning.

scholarly journals Identification of a unique filamin A binding region within the cytoplasmic domain of glycoprotein Ibα

2005 ◽  
Vol 387 (3) ◽  
pp. 849-858 ◽  
Author(s):  
Susan L. CRANMER ◽  
Inna PIKOVSKI ◽  
Pierre MANGIN ◽  
Philip E. THOMPSON ◽  
Teresa DOMAGALA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Von Willebrand Factor ◽  
Cytoplasmic Tail ◽  
Membrane Skeleton ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
Filamin A ◽  
Binding Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Binding of the platelet GPIb/V/IX (glycoprotein Ib/V/IX) receptor to von Willebrand factor is critical for platelet adhesion and aggregation under conditions of rapid blood flow. The adhesive function of GPIbα is regulated by its anchorage to the membrane skeleton through a specific interaction with filamin A. In the present study, we examined the amino acid residues within the cytoplasmic tail of GPIbα, which are critical for association with filamin A, using a series of 25-mer synthetic peptides that mimic the cytoplasmic tail sequences of wild-type and mutant forms of GPIbα. Peptide binding studies of purified human filamin A have demonstrated a major role for the conserved hydrophobic stretch L567FLWV571 in mediating this interaction. Progressive alanine substitutions of triple, double and single amino acid residues within the Pro561–Arg572 region suggested an important role for Trp570 and Phe568 in promoting GPIbα binding to filamin A. The importance of these two residues in promoting filamin A binding to GPIbα in vivo was confirmed from the study of Chinese-hamster ovary cells expressing GPIbα Trp570→Ala and Phe568→Ala substitutions. Phenotypic analysis of these cell lines in flow-based adhesion studies revealed a critical role for these residues in maintaining receptor anchorage to the membrane skeleton and in maintaining cell adhesion to a von Willebrand factor matrix under high-shear conditions. These studies demonstrate a novel filamin A binding motif in the cytoplasmic tail of GPIbα, which is critically dependent on both Trp570 and Phe568.

scholarly journals The Caenorhabditis elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor

1992 ◽  
Vol 287 (3) ◽  
pp. 995-999 ◽  
Author(s):  
S Ahmed ◽  
I N Maruyama ◽  
R Kozma ◽  
J Lee ◽  
S Brenner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Phorbol Ester ◽  
Catalytic Domain ◽  
Amino Acid Residues ◽  
C Elegans ◽  
High Affinity ◽  
Cell Extracts ◽  
Neuronal Connections ◽  
Phorbol Ester Receptor

The Caenorhabditis elegans unc-13 mutant is a member of a class of mutants that have un-coordinated movement. Mutations of the unc-13 gene cause diverse defects in C. elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system. unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family [Maruyama & Brenner (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5729-5733]. To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified. Phorbol ester binding to the 43 kDa protein was zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM). UNC-13 specific antisera detected a protein of approx. 190 kDa in wild-type (N2) but not in mutant (e1019) C. elegans cell extracts. We conclude that UNC-13 represents a novel class of phorbol ester receptor.

scholarly journals Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

2001 ◽  
Vol 67 (6) ◽  
pp. 2445-2452 ◽  
Author(s):  
Yuji Kannan ◽  
Yuichi Koga ◽  
Yohei Inoue ◽  
Mitsuru Haruki ◽  
Masahiro Takagi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Heat Inactivation ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon

ABSTRACT The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensissubtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding,T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly−82 to Gly316), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests thatT. kodakaraensis subtilisin exists in a monomeric form.T. kodakaraensis subtilisin hydrolyzed the synthetic substrateN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca2+ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.

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