Homology of kringle structures in urokinase and tissue-type plasminogen activator: The phylogeny with the related serine proteases.

Takahashi, Kei; Gojobori, Takashi; Naora, Hiroyuki

doi:10.1247/csf.10.209

Cited by 5 publications

(2 citation statements)

References 24 publications

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“…30). It is of particular interest to note here that the longest homologous segment, consisting of eleven residues (CQGDSGGPLVC) contains one enzyme-active site, the serine residue (7,22,28, and see below), and that this segment is located in a fragment (No.…”

Section: Resultsmentioning

confidence: 99%

A monoclonal antibody against human urokinase: The epitope structure and sequence homology with a human tissue-type plasminogen activator.

Takahashi

Naora

1985

Cell Struct. Funct.

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ABSTRACT. Our previous study showed that an epitope defined by a monoclonal antibody against human urokinase is located on the 33-Kdalton catalytic domain of the enzyme (Nakamura, M. et al., Cell Struct Funct., 9, 167-179, 1984). The epitope structure was further determined and characterized on one-dimensional SDS-polyacrylamide slab gel maps of CNBrcleaved polypeptide fragments as well as on their Western blots. A single homogeneous polypeptide with an approximate molecular weight of 3.4-Kdaltons was found to be antigenic. The monoclonal antibody exhibited a stronger inhibition of the enzyme activity than the polyclonal antibodies tested, and cross-reacted with a 65-Kdalton tissue-type plasminogen activator present in Detroit 562 cells. From these results and data made up with the help of a computer comparison of known sequences of urokinase and a tissue-type plasminogen activator, we concluded that the epitope is Cys-Gln-Gly-Asp-SerGly-Gly-Pro-Leu-Val-Cys and containes a catalytically active residue, serine.Plasminogen activators belong to a subgroup of serine-type proteases (21). The activators occur in multiple molecular weight forms (33, 35, 36) and play a prominent physiological role in the dissolution of thrombin (17). Among the activators, well studied ones are urokinase (7, 28) and a tissue-type activator (22); the molecular weights are around 54-and 65-Kdaltons, respectively. Salerno et al. (24) discovered a homogeneous mRNA of human kidney coding a single-chain pro-urokinase with an approximate molecular weight of 54-Kdaltons. Verde et al. (32) and Heyneker et al. (9) determined the full-length of nucleotide sequence of urokinase. Other lines of studies (1,14,19,29, 37) suggest that the 54-Kdalton enzyme is not a degradation product of a precursor molecule, although the de novo function of the enzyme is still not well understood.Recent studies on amino acid (7, 28) as well as nucleotide sequence (22) have made certain the molecular configuration of urokinase and the tissue-type activator and suggested that both enzymes are closely related. Of particular interest is that both have a characteristic preserved structure, kringle, which has no catalytic domain but Abbreviations used: MW, molecular weight; IgG, immunoglobulin G; SDS, sodium dodecylsulfate; PBS, phosphate buffered saline; BSA, bovine serum albumin; Kdalton, kilodalton; K1 and K2, two kringle structures present in a tissue-type plasminogen activator; FITC, fluorescein isothiocyanate; MEM, minimum essential medium. Enzyme, Urokinase (EC 3.4.21.31.) 195

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Section: Resultsmentioning

confidence: 99%

A monoclonal antibody against human urokinase: The epitope structure and sequence homology with a human tissue-type plasminogen activator.

Takahashi

Naora

1985

Cell Struct. Funct.

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“…Plasmin digests insoluble fibrin and thereby resolves the blood clot (Rijken et al, 1982). t-PA represents a multi domain protein and five domains can be distinguished: the N-terminal finger domain, the EGF (epidermal growth factor) domain, two kringle domains and the C-terminal protease domain (Banyai et al, 1983;Takahashi et al, 1985). Each domain plays a special role for the function of t-PA, in particular its interaction with a number of specific proteins.…”

Section: Introductionmentioning

confidence: 99%

Pentapeptide Identified as a Monoclonal Antibody Binding Site in the Serine-Protease Domain of t-PA

Gombert¹,

Werz²,

Schlüter³

et al. 1994

Biological Chemistry Hoppe-Seyler

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The first defined sequential epitope of the tissue plasminogen activator (t-PA) was determined by a monoclonal antibody against a synthetic peptide segment corresponding to peptide sequence 341-354 of t-PA. This segment was selected by computer assisted epitope prediction. Balb/c mice were immunized with catalase-peptide and tripalmitoyl-S-glyceryl-cys-teinyl-seryl-peptide conjugates. A monoclonal antibody derived from this immunization was reactive with native recombinant t-PA (rt-PA) and reduced carboxymethylated recombinant t-PA (RCM rt-PA). The sequential epitope was detected by Pepscan method using overlapping octa- and nonapeptides. By fine epitope mapping with tetra-, penta-, hexa- and heptapeptides the epitope was minimized to the pentapeptide EEEQK (347-351). Replacement set analysis confirmed the importance of this amino acid sequence, especially of the amino acid E348, for antibody binding. Functional assays of rt-PA were not affected by this antibody indicating that the epitope has no influence on the enzymatic center and the binding site of the inhibitor. The analysis demonstrates that the predicted recognition site of the monoclonal antibody 17-134/11 is exposed on the surface of the native rt-PA molecule.

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Identification and characterization of the epitopes of two monoclonal antibodies binding to the tissue-plasminogen activator rt-PA

Bayer¹,

Gombert²,

Werz³

et al. 1993

Peptides 1992

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Homology of kringle structures in urokinase and tissue-type plasminogen activator: The phylogeny with the related serine proteases.

Cited by 5 publications

References 24 publications

A monoclonal antibody against human urokinase: The epitope structure and sequence homology with a human tissue-type plasminogen activator.

A monoclonal antibody against human urokinase: The epitope structure and sequence homology with a human tissue-type plasminogen activator.

Pentapeptide Identified as a Monoclonal Antibody Binding Site in the Serine-Protease Domain of t-PA

Identification and characterization of the epitopes of two monoclonal antibodies binding to the tissue-plasminogen activator rt-PA

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