scholarly journals Identification and expression of Pen c 2, a novel allergen from Penicillium citrinum

1999 ◽  
Vol 341 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Lu-Ping CHOW ◽  
Ning-Yuan SU ◽  
Chia-Jung YU ◽  
Bor-Luen CHIANG ◽  
Horng-Der SHEN
Keyword(s):  
Amino Acid ◽  
Serine Proteases ◽  
Catalytic Triad ◽  
Penicillium Citrinum ◽  
E Coli ◽  
Acid Protein ◽  
Amino Acid Signal Peptide ◽  
Cloned Cdna ◽  
Allergic Disorders ◽  
Amino Acid Protein

The mould genus, Penicillium, is known to be a significant source of environmental aero-allergens. One important allergen from Penicillium citrinum, Pen c 2, has been identified by means of two-dimensional immunoblotting using IgE-containing patients' sera. This novel allergen was cloned, sequenced and expressed in Escherichia coli. The cloned cDNA encodes a large 457-amino acid protein precursor containing a 16-amino acid signal peptide, a 120-amino acid propeptide and the 321-amino acid mature protein. Comparison of the Pen c 2 sequence with known protein sequences revealed shared high sequence similarities with two vacuolar serine proteases from Aspergillus niger and Saccharomyces cerevisiae. Asp-46, His-78 and Ser-244 were found to constitute the catalytic triad of the 39-kDa Pen c 2. The DNA coding for Pen c 2 was cloned into vector PQE-30 and expressed in E. coli as a His-tag fusion protein that bound serum IgE from Penicillium-allergic patients on immunoblots. Recombinant Pen c 2 could therefore be used effectively for diagnosis and also potentially for the treatment of mould-derived allergic disorders.

scholarly journals Three novel paralogs of the rodent prolactin gene family

2000 ◽  
Vol 166 (1) ◽  
pp. 63-75 ◽  
Author(s):  
G Dai ◽  
D Wang ◽  
B Liu ◽  
JW Kasik ◽  
H Muller ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Family ◽  
Signal Peptide ◽  
Glycosylation Site ◽  
New Members ◽  
Acid Protein ◽  
Amino Acid Signal Peptide ◽  
Chorioallantoic Placenta ◽  
Amino Acid Protein ◽  
Amino Acid Signal

The prolactin (PRL) family consists of a collection of genes expressed in the uterus, placenta and anterior pituitary. These cytokines/hormones participate in the control of maternal-fetal adaptations to pregnancy. In this report, we establish the presence of three new members of the PRL family. Novel expressed sequence tags (ESTs) with homology to PRL were isolated from embryonic and placental cDNA libraries. The cDNAs were sequenced and compared with those of other members of the PRL family. The three new cDNAs were assigned to the PRL family on the basis of sequence similarities and were referred to as PRL-like protein-J (PLP-J), PRL-like protein-K (PLP-K) and PRL-like protein-M (PLP-M). Both rat and mouse PLP-J cDNAs were identified. Rat PLP-J cDNA encodes for a predicted 211 amino acid protein containing a 29 amino acid signal peptide and two putative N-linked glycosylation sites, whereas the mouse PLP-J cDNA encodes for a 212 amino acid protein containing a 29 amino acid signal peptide with a single N-linked glycosylation site. Rat and mouse PLP-J proteins share approximately 79% and 70% nucleotide and amino acid sequence identity, respectively. A full-length rat PLP-K cDNA and a partial tentative mouse PLP-K cDNA were identified. The rat PLP-K cDNA encodes for a predicted 228 amino acid protein containing a 31 amino acid signal peptide and one putative N-linked glycosylation site; the mouse PLP-M cDNA encodes for a predicted 228 amino acid protein containing a 28 amino acid signal peptide and one putative N-linked glycosylation site. Genes for PLP-J, PLP-K and PLP-M are situated at the Prl family locus on mouse chromosome 13. PLP-J was exclusively expressed in decidual tissue from both the mouse and rat. PLP-K was expressed in trophoblast cells of the chorioallantoic placenta and showed an apparent species difference. In the mouse, virtually all trophoblast lineages expressed PLP-K, whereas in the rat, PLP-K expression was restricted to the labyrinthine trophoblast cells. Mouse PLP-M expression was restricted to the junctional zone of the chorioallantoic placenta. In summary, we have identified three new members of the rodent PRL gene family that are expressed in uterine and placental structures. Future experimentation is needed to determine the specific roles of each of these ligands in the biology of pregnancy.

scholarly journals Molecular and immunological characterization and IgE epitope mapping of Pen n 18, a major allergen of Penicillium notatum

2002 ◽  
Vol 363 (3) ◽  
pp. 707-715 ◽  
Author(s):  
Chia-Jung YU ◽  
Yen-Ming CHEN ◽  
Song-Nan SU ◽  
Farhad FOROUHAR ◽  
Shu-Hua LEE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Serine Proteases ◽  
Major Allergen ◽  
Penicillium Notatum ◽  
Ige Binding ◽  
Acid Protein ◽  
Binding Epitope ◽  
Cross Inhibition ◽  
Amino Acid Protein

The mould genus, Penicillium, is a significant source of environmental aero-allergens. A major allergen from Penicillium notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of Penicillium citrinum, followed by matrix-assisted laser-desorption ionization—time-of-flight MS analysis of the peptide digest. Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence. The cDNA encoded a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N- and C-terminal prosequences. The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi. The Pen n 18 coding sequence was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni2+-chelate affinity chromatography. On immunoblots, the purified recombinant protein specifically bound IgE from mould-allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P. citrinum, Pen c 18. When mapping of the allergenic epitopes was performed, at least nine different linear IgE-binding epitopes, located throughout the Pen n 18 protein, were identified. Of these, peptide C12, located in the N-terminal region of the molecule, was recognized by serum from 75% of the patients tested and therefore appears to be an immunodominant IgE-binding epitope.

scholarly journals New lnu(C) Gene Conferring Resistance to Lincomycin by Nucleotidylation in Streptococcus agalactiae UCN36

2005 ◽  
Vol 49 (7) ◽  
pp. 2716-2719 ◽  
Author(s):  
Adeline Achard ◽  
Corinne Villers ◽  
Vianney Pichereau ◽  
Roland Leclercq
Keyword(s):  
Amino Acid ◽  
Streptococcus Agalactiae ◽  
Open Reading Frames ◽  
Inverted Repeats ◽  
E Coli ◽  
Acid Protein ◽  
Total Dna ◽  
Plasmid Puc18 ◽  
Reading Frames ◽  
Amino Acid Protein

ABSTRACT Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 μg/ml) but susceptible to clindamycin (MIC = 0.12 μg/ml) and erythromycin (MIC = 0.06 μg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS1. The downstream ORF, lnu(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuAN2, LnuA, and LnuA′ lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus, respectively. Crude lysates of E. coli AG100A containing the cloned lnu(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl2. Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin.

scholarly journals T-SP1: a novel serine protease-like protein predominantly expressed in testis

2008 ◽  
Vol 389 (12) ◽  
Author(s):  
Peter Neth ◽  
Birgit Profanter ◽  
Claudia Geissler ◽  
Dorit K. Nägler ◽  
Andreas Nerlich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Serine Protease ◽  
Serine Proteases ◽  
Coagulation Factor ◽  
Catalytic Triad ◽  
Chromosome 8 ◽  
C Terminus ◽  
Amino Acid Signal Peptide ◽  
Rapid Amplification ◽  
Testis Tissue

Abstract Here, we describe a novel member in the group of membrane-anchored chymotrypsin (S1)-like serine proteases, namely testis serine protease 1 (T-SP1), as it is principally expressed in testis tissue. The human T-SP1 gene encompasses 28.7 kb on the short arm of chromosome 8 and consists of seven exons. Rapid amplification of cDNA ends (RACE) experiments revealed that due to alternative splicing three different variants (T-SP1/1, -2, -3) are detectable in testis tissue displaying pronounced heterogeneity at their 3′-end. T-SP1/1 consists of an 18 amino acid signal peptide and of a 49 amino acid propeptide. The following domain with the catalytic triad of His108, Asp156, and Ser250 shares sequence identities of 42% and 40% with the blood coagulation factor XI and plasma kallikrein, respectively. Only T-SP1/1 contains a hydrophobic part at the C-terminus, which provides the basis for cell membrane anchoring. Using a newly generated polyclonal anti-T-SP1 antibody, expression of the T-SP1 protein was found in the Leydig and Sertoli cells of the testis and in the epithelial cells of the ductuli efferentes. Notably, T-SP1 protein was also detectable in prostate cancer and in some ovarian cancer tissues, indicating tumor-related synthesis of T-SP1 beyond testis tissue.

scholarly journals Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (15) ◽  
pp. 5067-5074 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Ring Cleavage ◽  
Sphingomonas Paucimobilis ◽  
Acid Protein ◽  
Demethylation Pathway ◽  
Dioxygenase Gene ◽  
Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

scholarly journals LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

2006 ◽  
Vol 398 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Yukiko Mizutani ◽  
Akio Kihara ◽  
Yasuyuki Igarashi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Family Members ◽  
Fatty Acyl ◽  
Ceramide Synthase ◽  
Broad Substrate Specificity ◽  
Acid Protein ◽  
Amino Acid Protein

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

scholarly journals Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

1998 ◽  
Vol 64 (5) ◽  
pp. 1650-1656 ◽  
Author(s):  
Peter W. Coschigano ◽  
Thomas S. Wehrman ◽  
L. Y. Young
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Molecular Mass ◽  
Cysteine Residue ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Pyruvate Formate Lyase ◽  
Calculated Molecular Mass ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

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