scholarly journals Sequence of bovine carbonic anhydrase VI: potential recognition sites for N-acetylgalactosaminyltransferase

1996 ◽  
Vol 318 (1) ◽  
pp. 291-296 ◽  
Author(s):  
Weiping JIANG ◽  
Joseph T WOITACH ◽  
Dwijendra GUPTA
Keyword(s):  
Amino Acids ◽  
Submaxillary Gland ◽  
Cdna Clones ◽  
Carbonic Anhydrases ◽  
Active Site Residues ◽  
Glycoprotein Hormone ◽  
Bovine Carbonic Anhydrase ◽  
Glycosylation Sites ◽  
Carbonic Anhydrase Vi ◽  
Related Proteins

Carbonic anhydrases (CAs I–VII) are products of a gene family that encodes seven isoenzymes and several CA-related proteins. We report the cloning and sequencing of the cDNA clones encoding one of these isoenzymes, CA VI, from bovine submaxillary gland. The translated polypeptide consists of 319 amino acids, including a signal peptide (14 amino acids) typical of secreted proteins. The predicted mature protein contains 305 amino acids including a 13-amino-acid C-terminal sequence that is also present in the sheep but absent in human CA VI. The deduced mature bovine protein is 87% and 68% identical to that of sheep and human CA VI, respectively. Active-site residues of the enzyme, as well as the three zinc-binding histidines and the two cysteines involved in an intra-chain disulphide bond, are all conserved in the three species. Two potential Asn-glycosylation sites are also conserved, both of which appear to be glycosylated in sheep and bovine CA VI. Two potential peptide recognition sequences are present in bovine CA VI for the glycoprotein hormone: N-acetylgalactosaminyltransferase (GalNAc-transferase), which is one of the two transferases required to form GalNAc-4-SO4 in bovine CA VI-linked oligosaccharides. Specifically, these two sequences are Asp-Leu-Lys-Met-Lys-Lys and Ile-Thr-Lys-Arg-Lys-Lys. Comparison of these sequences with sheep and human CA VI sequences indicates that distinct glycoforms of CA VI could exist in submaxillary gland from different species.

scholarly journals A cDNA clone for human glucosamine-6-sulphatase reveals differences between arylsulphatases and non-arylsulphatases

1992 ◽  
Vol 288 (2) ◽  
pp. 539-544 ◽  
Author(s):  
D A Robertson ◽  
C Freeman ◽  
C P Morris ◽  
J J Hopwood
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Similarity ◽  
Genetic Disorder ◽  
Heparan Sulphate ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Lysosomal Storage ◽  
Glycosylation Sites

Glucosamine-6-sulphatase is an exo-hydrolase required for the lysosomal degradation of heparan sulphate and keratan sulphate. Deficiency of glucosamine-6-sulphatase activity leads to the lysosomal storage of the glycosaminoglycan, heparan sulphate and the monosaccharide sulphate N-acetylglucosamine 6-sulphate and the autosomal recessive genetic disorder mucopolysaccharidosis type IIID. Glucosamine-6-sulphatase can be classified as a non-arylsulphatase since, relative to arylsulphatase B, it shows negligible activity toward 4-methylumbelliferyl sulphate. We have isolated human cDNA clones and derived amino acid sequence coding for the entire glucosamine-6-sulphatase protein. The predicted sequence has 552 amino acids with a leader peptide of 36 amino acids and contains 13 potential N-glycosylation sites, of which it is likely that 10 are used. Glucosamine-6-sulphatase shows strong sequence similarity to other sulphatases such as the family of arylsulphatases, although the degree of similarity is not as high as that between members of the arylsulphatase family. This pattern of inter- and intra-family similarity delineates regions and amino acid residues that may be critical for sulphatase function and substrate specificity.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

scholarly journals The Superagonistic Activity of Bovine Thyroid-stimulating Hormone (TSH) and the Human TR1401 TSH Analog Is Determined by Specific Amino Acids in the Hinge Region of the Human TSH Receptor

2009 ◽  
Vol 284 (24) ◽  
pp. 16317-16324 ◽  
Author(s):  
Sandra Mueller ◽  
Gunnar Kleinau ◽  
Mariusz W. Szkudlinski ◽  
Holger Jaeschke ◽  
Gerd Krause ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thyroid Stimulating Hormone ◽  
Hinge Region ◽  
Camp Production ◽  
Glycoprotein Hormone ◽  
Charged Amino Acids ◽  
Bovine Thyroid ◽  
Positively Charged ◽  
Bovine Tsh ◽  
Signaling Activity

Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu297, Glu303, and Asp382) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu297, Glu303, or Asp382 and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp382 seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.

scholarly journals Cloning and sequence analysis of cDNAs encoding the α1, α2 and α3 chains of mouse collagen VI

1993 ◽  
Vol 291 (3) ◽  
pp. 787-792 ◽  
Author(s):  
R Z Zhang ◽  
T C Pan ◽  
R Timpl ◽  
M L Chu
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Untranslated Regions ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Collagen Vi ◽  
Central Segment ◽  
Glycosylation Sites ◽  
Key Features ◽  
Triple Helical Domain

cDNA clones encoding the alpha 1, alpha 2 and alpha 3 chains of mouse collagen VI have been isolated by screening cDNA libraries with the corresponding human probes. The composite cDNAs for the alpha 1, alpha 2, and alpha 3 chains are 2.5, 1.6 and 2.9 kb in size respectively. The alpha 1 and alpha 2 cDNAs encode the C-terminal portions of the chains as well as the entire 3′-untranslated regions, while the alpha 3 cDNAs encode a central segment of 959 amino acids flanking the triple-helical domain. The deduced amino acid sequences share 86-88% identity with the human counterparts and 67-73% identity with the chicken equivalents. Alignment of the deduced amino acid sequences of mouse, human and chicken collagens reveal that the key features of the protein, including the cysteine residues, imperfections in the Gly-Xaa-Xaa regions, Arg-Gly-Asp sequences and potential N-glycosylation sites, are mostly conserved.

scholarly journals Identification and characterization of a novel cytoskeleton-associated pp60src substrate.

1991 ◽  
Vol 11 (10) ◽  
pp. 5113-5124 ◽  
Author(s):  
H Wu ◽  
A B Reynolds ◽  
S B Kanner ◽  
R R Vines ◽  
J T Parsons
Keyword(s):  
Cell Structure ◽  
Transformed Cells ◽  
Cdna Clones ◽  
Amino Terminal ◽  
Multidomain Structure ◽  
Striking Change ◽  
Carboxyl Terminal ◽  
Amino Terminal Domain ◽  
Related Proteins

Transformation of cells by the src oncogene results in elevated tyrosine phosphorylation of two related proteins, p80 and p85 (p80/85). Immunostaining with specific monoclonal antibodies revealed a striking change of subcellular localization of p80/85 in src-transformed cells. p80/85 colocalizes with F-actin in peripheral extensions of normal cells and rosettes (podosomes) of src-transformed cells. Sequence analysis of cDNA clones encoding p80/85 revealed an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids, a carboxyl-terminal SH3 domain, and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The multidomain structure of p80/85 and its colocalization with F-actin in normal and src-transformed cells suggest that these proteins may associate with components of the cytoskeleton and contribute to organization of cell structure.

scholarly journals Molecular cloning and characterization of ornithine decarboxylase cDNA of the nematode Panagrellus redivivus

1995 ◽  
Vol 308 (2) ◽  
pp. 635-640 ◽  
Author(s):  
H von Besser ◽  
G Niemann ◽  
B Domdey ◽  
R D Walter
Keyword(s):  
Amino Acids ◽  
Active Sites ◽  
Cdna Clones ◽  
Degenerate Primers ◽  
Calculated Molecular Mass ◽  
Panagrellus Redivivus ◽  
Free Living Nematode ◽  
Mixed Stage ◽  
Conserved Amino Acids

In a PCR with degenerate primers encoding highly conserved amino acids within ornithine decarboxylases (ODCs) of several organisms, a fragment of the ODC gene of the free-living nematode Panagrellus redivivus was isolated. Northern blot analysis revealed a single 1.7 kb transcript in a mixed-stage population of animals. From this RNA source, a cDNA library was constructed and screened with the PCR fragment. Several cDNA clones were isolated, one of which encodes the complete 435-amino-acid ODC enzyme with a calculated molecular mass of 47.1 kDa. The P. redivivus ODC possesses 126 of the 136 highly conserved amino acids in the enzymes from fungi, invertebrates and vertebrates. Functional amino acids are conserved, suggesting that the two active sites of the P. redivivus ODC are formed at the interface of a homodimer, as described for mammalian ODCs.

The efficiency of N-linked glycosylation of bovine DNase I depends on the Asn-Xaa-Ser/Thr sequence and the tissue of origin

2001 ◽  
Vol 355 (1) ◽  
pp. 245-248 ◽  
Author(s):  
Atsushi NISHIKAWA ◽  
Sumi MIZUNO
Keyword(s):  
Submaxillary Gland ◽  
Dnase I ◽  
The State ◽  
Sugar Chain ◽  
Glycosylation Sites ◽  
Tissue Of Origin ◽  
Sugar Chains

Bovine DNase I contains two potential N-linked glycosylation sites with the sequences Asn18-Ala-Thr and Asn106-Asp-Ser. A previous report established that pancreatic DNase I has only one sugar chain at Asn18 [Liao, Salnikow, Moore and Stein (1973) J. Biol. Chem. 248, 1489–1495]. We found, however, that bovine DNase I expressed in COS-1 cells was glycosylated about 70% at Asn106 in addition to being completely glycosylated at Asn18. Glycosylation of Asn106 increased to 97% when Asp107 was mutated to Glu or when Ser108 was mutated to Thr. Mutation of Asp107 to Trp had no effect, whereas a substitution with Pro at this position abolished glycosylation of Asn106. Analysis of the state of glycosylation of DNase I purified from a variety of bovine tissues revealed that DNase I from spleen, submaxillary gland, lung and adrenal had two sugar chains, whereas enzyme from pancreas and kidney had only one sugar chain. These findings demonstrate a major difference in the ability of various tissues to utilize N-linked glycosylation signals that contain suboptimal residues in the second and third positions.

Characterization and expression of the human rhoH12 gene product

1989 ◽  
Vol 9 (5) ◽  
pp. 2058-2066
Author(s):  
H Avraham ◽  
R A Weinberg
Keyword(s):  
Amino Acids ◽  
Cell Lines ◽  
Soft Agar ◽  
Cdna Clones ◽  
Focus Formation ◽  
Reductase Gene ◽  
Evolutionarily Conserved ◽  
Normal Human ◽  
Nih 3T3 ◽  
P21 Proteins

The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in place of the glycine and leucine in place of the glutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via cotransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho protein was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.

Pax-6, a murine paired box gene, is expressed in the developing CNS

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1435-1449 ◽  
Author(s):  
C. Walther ◽  
P. Gruss
Keyword(s):  
Amino Acids ◽  
Neural Tube ◽  
Multigene Family ◽  
Ventricular Zone ◽  
Open Reading Frame ◽  
Cdna Clones ◽  
Coding Region ◽  
Paired Domain ◽  
Reading Frame ◽  
Pax Genes

A multigene family of paired-box-containing genes (Pax genes) has been identified in the mouse. In this report, we describe the expression pattern of Pax-6 during embryogenesis and the isolation of cDNA clones spanning the entire coding region. The Pax-6 protein consists of 422 amino acids as deduced from the longest open reading frame and contains, in addition to the paired domain, a paired-type homeodomain. Beginning with day 8 of gestation, Pax-6 is expressed in discrete regions of the forebrain and the hindbrain. In the neural tube, expression is mainly confined to mitotic active cells in the ventral ventricular zone along the entire anteroposterior axis starting at day 8.5 of development. Pax-6 is also expressed in the developing eye, the pituitary and the nasal epithelium.

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not

1987 ◽  
Vol 7 (6) ◽  
pp. 2173-2179
Author(s):  
P C Yelick ◽  
R Balhorn ◽  
P A Johnson ◽  
M Corzett ◽  
J A Mazrimas ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Protamine 1 ◽  
Protamine 2

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.

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