Monoclonal antibodies to human urokinase identify the single-chain pro-urokinase precursor.

Salerno, Grazia Gabriella; Verde, Pasquale; Nolli, Maria Luisa; Corti, Angelo; Szöts, Hannelore; Meo, Tommaso; Johnson, Julie; Bullock, Sarah; Cassani, Giovanni; Blasi, Francesco

doi:10.1073/pnas.81.1.110

Cited by 36 publications

(19 citation statements)

References 26 publications

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“…Several monoclonal antibodies to human urokinase have been produced recently (6,12,29,39). The site of the specific cells in kidney that synthesize and secrete the antigen is not yet known.…”

Section: Discussionmentioning

confidence: 99%

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A monoclonal antibody against human urokinase: Characterization of the epitope and its localization in human kidney.

Nakamura

Takahashi

Naora

et al. 1984

Cell Struct. Funct.

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ABSTRACT. Monoclonal antibodies against human plasminogen activator urokinase have been produced. A G62 hybridoma-producing antibody (IgG) was purified on a DEAE-cellulose column, and it proved useful for the measurement, identification and purification of antigens that had approximate molecular weights of 55-and 33-Kdaltons.For immunochemical measurements and purification, a competitive enzymelinked immunosorbent assay (ELISA) and affinity chromatography using antibody-immobilized Sepharose 4B were developed. The ELISA has sensitivity to 20 p mole antigen molecules. The binding capacity of the antigen on the affinity column was evaluated on SDS-polyacrylamide slab gels as well as by fibrin autography and ELISA. Results showed that there was quantitative purification with no loss of enzyme activity in the one-step procedure.Western blotting and affinity binding showed antigenic bands with apparent molecular weights of 55-and 33-Kdaltons. Because the 55-Kdalton form contains 33-and 22-Kdalton components connected by a disulfide bond, the epitope domain is present on the 33-Kdalton chain.Using this antibody, we examined human kidney sections by direct immunofluorescence to locate the antigen. It was found in epithelial cells of convoluted segments, in glomerulus cells and in capillary endothelial cells, evidence that renal tubular cells synthesize the antigen which then is secreted in urine.

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Section: Discussionmentioning

confidence: 99%

“…Recently, the induction of a large amount of the urokinase was demonstrated in DNA-repair-deficient cells that had undergone UV irradiation (5,21). Human urokinase is synthesized as a single-chain proenzyme in the kidney and probably is secreted in urine (4,29,34). The amino acid sequences and structure of this enzyme have been well studied (8,9,25,33,39).…”

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confidence: 99%

A monoclonal antibody against human urokinase: Characterization of the epitope and its localization in human kidney.

Nakamura

Takahashi

Naora

et al. 1984

Cell Struct. Funct.

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“…Plasminogen actix ator is a serine protease existing in two forms known as tissue type (tPA) and urokinase tx-pe (uPA) (Sobel et al 1952: Giinzler et al 1982a.b: Nielsen et al 1982: Wun et al 1982: Salerno et al 1984: Riccio et al 1985. uPA converts plasminogen into plasmin and thus mediates pericellular proteolysis during cell migration and tissue remodelling, under phy siological and pathophy siological conditions (Gnrmann et al 1976: Gerdin and Saldeen.…”

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confidence: 99%

In vitro inhibition of human malignant brain tumour cell line proliferation by anti-urokinase-type plasminogen activator monoclonal antibodies

Abaza

Shaban²,

Narayan³

et al. 1998

Br J Cancer

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Summary A brain tumour-associated marker, urokinase (UK), was investigated using rabbit anti-UK polyclonal and munne anti-UK monoclonal antibodies, which were prepared by immunization with low molecular weight UK (LMW-UK) and high molecular weight urokinase (HMW-UK) synthetic peptide respectively. The polyclonal antibody cross-reacted with both LMW-UK and HMW-UK. whereas the murne MAbs were specific for HMW-UK. These immunological probes were used to study urokinase in glioma extracts, tissues, sera and cell lines that had been prepared from prmary cultures of freshly dissected gliomas. Radioimmunoassays showed that glioma extracts had much higher level (5-to 44-fold) of UK than normal human brain extracts. This result was confirmed by immunoblotting of electrophoresis gels of glioma and human brain extracts. Immunohistochemical study using anti-UK MAb demonstrated much higher levels of UK in glioma tissue than normal brain tissue. Immunohistochemical study using anti-UK MAbs localized UK on the cell surface of glioma cells. Anti-UK MAbs inhibited the proliferation of AA cell lines and GB cell lines (500c to > 90%) and exerted minor effects (< 20%) on normal human liver, intestine and lymphocyte cell lines. Taken together, these results suggest that anti-UK MAbs may have therapeutic potential for human gliomas and cancer metastasis.Keywords: glioma; cell line: anti-UK-MAb; expression: surface location: anti-proliferative activity Considerable indirect ex idence from model tumour svstems has accumulated to show that invasion and metastasis in solid tumours require the action of tumour-associated proteases that promote the dissolution of the surrounding tumour matrix and the basement membranes. Receptor-bound urokinase-txpe plasminogen actixvator (uPA) appears to play a key role in these ex ents (Dano et al. 1986: Duffx. 1987and Zucker. 1988 Correspondence to M-SI Abaza membrane. Hence. receptor-bound uPA v ill promote plasminogen actixation and thus the dissolution of the tumour matrix and the basement membrane. which is a prerequisite for invasion and metastasis.Plasminogen actix ators in tumours of the central nernous sx stem hax e not been studied extensix ely despite the intense in estigation into their possible role in cancer biologv Hoosein et al. 1991: Foekens et al. 1992: Hollas et al. 1992: Kobaxashi et al. 1992 Reith and Rucklidge et al. 1992: Sumivoshi et al. 1992: Hsui et al. 1993: Janicke et al. 1993: Pujade-Lauraine et al. 1993: Yamashita et al. 1993: Achbarou et al. 1994: Bianchi et al. 1994: Bouchet et al. 1994and Young et al. 1994. In this study. we report the oxverexpression of urokinasetype plasminogen activator in -liomas. its localization on human glioma cell surface and the antitumorigenic effect of anti-UK MAbs against human malignant brain tumour cell lines. MATERIALS AND METHODS MaterialsBrain tumour extracts were prepared from wet tissue samples (2 g) homocenized by biohomogenizer (Fisher Scientific) in 10 mxis phosphate-buffered saline (PBS) buffer (pH 7.2) at 4 C wxith 100 nvm pheny...

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“…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.…”

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confidence: 99%

“…To investigate further the functional role of enzymes which might have more stringent and distinct immunological properties, several authors (8,11,19,24,34) have produced monoclonal antibodies against urokinase. The usefulness of monoclonal antibodies for the quantification, purification, and characterization of the antigen has been described by us (19) and others (8,34).…”

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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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Monoclonal antibodies to human urokinase identify the single-chain pro-urokinase precursor.

Cited by 36 publications

References 26 publications

A monoclonal antibody against human urokinase: Characterization of the epitope and its localization in human kidney.

A monoclonal antibody against human urokinase: Characterization of the epitope and its localization in human kidney.

In vitro inhibition of human malignant brain tumour cell line proliferation by anti-urokinase-type plasminogen activator monoclonal antibodies

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

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