Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase.

LaVallie, Edward R.; Rehemtulla, Alnawaz; Racie, L.A.; Diblasio, Elizabeth; Ferenz, Catherine R.; Grant, Kathleen L.; Light, Albert; McCoy, John

doi:10.1016/s0021-9258(19)49464-7

Cited by 108 publications

(23 citation statements)

References 51 publications

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“…Fc chimeras were purified by protein A-Sepharose (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) chromatography per the manufacturer's instructions and quantified by an anti-Fc enzyme-linked immunosorbent assay using human IgG 1 (Sigma Chemical Co.) as a standard. The 19.Fc form of PSGL-1 described earlier (44) was mutated to include an enterokinase cleavage site (19) located between the PSGL-1 and IgG Fc regions and is termed 19.ek.Fc. A plasmid encoding this construct was stably transfected into CHO cells that were engineered to also express Fuc-TVII and ␤ (1,6)-N -acetylglucosaminyltransferase ("core2" [9]) activities (17,25).…”

Section: Soluble Forms Of Psgl-1mentioning

confidence: 99%

“…A plasmid encoding this construct was stably transfected into CHO cells that were engineered to also express Fuc-TVII and ␤ (1,6)-N -acetylglucosaminyltransferase ("core2" [9]) activities (17,25). 19…”

Section: Soluble Forms Of Psgl-1mentioning

confidence: 99%

“…The second construct, 19.ek.Fc, is a modified version of the 19.Fc construct used in the earlier study (44). 19.ek.Fc contains only the first 19 amino acids of PSGL-1, which includes the anionic, sulfated tyrosine region and a critical O-linked glycosylation site at Threonine-16, Thr-16, also present in the 148.Fc. Unique to the 19.ek.Fc construct is a cleavable polypeptide sequence introduced between the PSGL-1 and Fc sequences.…”

Section: Psgl-1 Regions Necessary And/or Sufficient For Attachment and Rolling On Cho-e And -P Monolayersmentioning

confidence: 99%

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Isolated P-selectin Glycoprotein Ligand-1 Dynamic Adhesion to P- and E-selectin

Goetz

Greif

Ding

et al. 1997

The Journal of Cell Biology

136

131

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Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P-and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-μm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-α–activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E-or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x–containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1–mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.

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Section: Soluble Forms Of Psgl-1mentioning

confidence: 99%

Section: Soluble Forms Of Psgl-1mentioning

confidence: 99%

Section: Psgl-1 Regions Necessary And/or Sufficient For Attachment and Rolling On Cho-e And -P Monolayersmentioning

confidence: 99%

See 1 more Smart Citation

Isolated P-selectin Glycoprotein Ligand-1 Dynamic Adhesion to P- and E-selectin

Goetz

Greif

Ding

et al. 1997

The Journal of Cell Biology

136

131

View full text Add to dashboard Cite

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“…These reports consider two families with affected siblings (Tarlow et al 1970;Haworth et al 1975), including one family with affected individuals of either gender, a finding suggesting autosomal recessive inheritance (Haworth et al 1975). On the basis of the isolation of a partial bovine enteropeptidase cDNA (LaVallie et al 1993), Kitamoto et al (1994Kitamoto et al ( , 1995 cloned the cDNAs containing the complete coding regions of bovine and human proenteropeptidase and mapped the human gene to chromosome 21q21, by FISH. According to the deduced amino acid sequence, enteropeptidase is a serine protease.…”

Section: Introductionmentioning

confidence: 99%

Mutations in the Proenteropeptidase Gene Are the Molecular Cause of Congenital Enteropeptidase Deficiency

Holzinger

Maier

Bück

et al. 2002

The American Journal of Human Genetics

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Enteropeptidase (enterokinase [E.C.3.4.21.9]) is a serine protease of the intestinal brush border in the proximal small intestine. It activates the pancreatic proenzyme trypsinogen, which, in turn, releases active digestive enzymes from their inactive pancreatic precursors. Congenital enteropeptidase deficiency is a rare recessively inherited disorder leading, in affected infants, to severe failure to thrive. The genomic structure of the proenteropeptidase gene (25 exons, total gene size 88 kb) was characterized in order to perform DNA sequencing in three clinically and biochemically proved patients with congenital enteropeptidase deficiency who were from two families. We found compound heterozygosity for nonsense mutations (S712X/R857X) in two affected siblings and found compound heterozygosity for a nonsense mutation (Q261X) and a frameshift mutation (FsQ902) in the third patient. In accordance with the biochemical findings, all four defective alleles identified are predicted null alleles leading to a gene product not containing the active site of the enzyme. These data provide first evidence that proenteropeptidase-gene mutations are the primary cause of congenital enteropeptidase deficiency.

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“…Peptides or proteins bearing an N-terminal cysteine can be prepared either synthetically or by proteolytic cleavage of recombinant protein carrying an engineered site. 20 The 5'terminal phosphorothioate moiety can be easily introduced into ribo-and deoxyribo-nucleic acids either chemically 21 or enzymatically. 22 The ligation proceeds in good yield and the strategy is compatible with conditions 23 employed in the ligation of peptide fragments containing mutiple cysteine residues.…”

mentioning

confidence: 99%

Synthesis of an RNA-Peptide Conjugate by Orthogonal Ligation

McPherson¹,

Wright²

1999

Synlett

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A convergent strategy for the synthesis of an RNApeptide conjugate is presented. Regioselective ligation between a 5'-modified RNA harboring a 5'-thioester group and a peptide carrying an N-terminal cysteine afforded an RNA-peptide conjugate under physiological conditions without the need for protecting groups.We have been interested in the synthesis of covalent nucleic acid-peptide conjugates for the construction of encoded peptide libraries. 1 In particular, we wanted to develop a convergent ligation strategy for the synthesis of RNA-protein conjugates. Ideally, this ligation reaction should take place between the native protein and the RNA with minimal chemical manipulation and under physiological conditions. A review of the literature revealed several step-wise synthetic schemes using protecting group strategies to prepare oligonucleotide-peptide conjugates. 2 Alternatively, convergent schemes have been devised for attaching unprotected peptides to oligonucleotides using a covalent linkage, such as disulfide, 3 thioether, 4 amide 5,6 or by Schiff base formation between an e-amino group of lysine and an oligonucleotide 2'-(3')-dialdehyde. 7 Analogous schemes have been developed for attaching RNA to proteins. 8 A major drawback in using protein thiols or amines for conjugation is the lack of regiospecific ligation upon reaction with proteins containing multiple cysteine or lysine residues.We now report a highly regiospecific method for the preparation of RNA-peptide conjugates. Our strategy is based on orthogonal ligation chemistry, which has proven highly successful in the ligation of large, unprotected peptide fragments in solution. 9,10 Chemoselective ligation between a 5'-modified RNA harboring a 5'-thioester group and a polypeptide bearing a cysteine residue at the amino terminus yields a well defined 5'-RNA-peptide conjugate (Scheme). Ligation is initiated via a transthioesterification reaction followed by a spontaneous intramolecular rearrangement to form an amide bond between the RNA and the peptide.We initially developed a simplified model to study the ligation chemistry (Figure 1). Guanosine-5'-phosphorothioate 1 (GMPS) was used as a mimic for RNA carrying a 5'-phosphorothioate group. A phenyl thioester group was introduced via S-alkylation of the phosphorothioate moiety of 1 with phenyl a-bromothioacetate 2. 11 Alkylation of 1 with an excess of 2 was quantitative and complete in 5 min as monitored by reversed phase HPLC. 12 A single product 3 was observed and alkylation of the phosphorothioate group was confirmed by 31 P NMR. 13 No N-or O-alkylated 1 was detected ( 1 H NMR, MS) after prolonged reaction times (1h). The product 3 was then treated with a stoichiometric amount of Cys-Gly and analysis of the reaction mixture by RP-HPLC showed the disappearance of 3 (R t = 14.8 min) and the formation of a new peak at R t = 11.2 min (see HPLC chromatogram, Figure 1). 14 The structure of the nucleotide-dipeptide conjugate 4 was confirmed by NMR and mass spectroscopy. 15 Following efficient ligation in our...

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Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase.

Cited by 108 publications

References 51 publications

Isolated P-selectin Glycoprotein Ligand-1 Dynamic Adhesion to P- and E-selectin

Isolated P-selectin Glycoprotein Ligand-1 Dynamic Adhesion to P- and E-selectin

Mutations in the Proenteropeptidase Gene Are the Molecular Cause of Congenital Enteropeptidase Deficiency

Synthesis of an RNA-Peptide Conjugate by Orthogonal Ligation

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