Differential proteolysis and evidence for a residue exchange in tissue plasminogen activator suggest possible association between two types of protein microheterogeneity

Jörnvall, Hans; Pohl, Gunnar; Bergsdorf, Nils; Wallén, Per

doi:10.1016/0014-5793(83)80245-2

Cited by 50 publications

(13 citation statements)

References 13 publications

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“…The extension of this "pro"-segment cannot be determined because of the NH2-terminal heterogeneity of the mature t-PA protein (39). Sequence analysis of purified t-PA from melanoma cells has shown that the protein starts at the serine designated number 1 or at the glycine designated number -3 ( Fig.…”

Section: Correlation Of Exon Regions With Structural Units Of Thementioning

confidence: 99%

“…Assuming that the longer form of t-PA is the cleaved product, these amino acids may be part of a recognition sequence for the enzyme system(s) that cleaves "pro"-sequences (40). The shorter form of t-PA may result from additional proteolytic digestion, presumably by a plasmin-like enzyme in the culture medium (39). In the t-PA gene, the signal peptide and the "pro"-segment are coded by separate exons, whereas both of these segments belong to the same exon in the serum albumin gene (41).…”

Section: Correlation Of Exon Regions With Structural Units Of Thementioning

confidence: 99%

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The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains.

Ny¹,

Elgh²,

Lund³

1984

Proc. Natl. Acad. Sci. U.S.A.

275

109

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A genomic clone carrying the human tissuetype plasminogen activator (t-PA) gene was isolated from a cosmid library, and the gene structure was elucidated by restriction mapping, Southern blotting, and DNA sequencing. The cosmid contained all the coding parts of the mRNA, except for the first 58 bases in the 5' end of the mRNA, and had a total length of >20 kilobases. It was separated into at least 14 exons by at least 13 introns, and the exons seemed to code for structural or functional domains. Thus, the signal peptide, the propeptide, and the domains of the heavy chain, including the regions homologous to growth factors, and to the "finger" structure of fibronectin, are all encoded by separate exons. In addition, the two kringle regions of t-PA were both coded for by two exons and were cleaved by introns at identical positions. The region coding for the light chain, comprising the serine protease part of the molecule was split by four introns, revealing a gene organization similar to other serine proteases.Plasminogen activators (PAs), are a class of serine proteases that convert the proenzyme plasminogen into plasmin, which then degrades the fibrin network of blood clots (1, 2). The PAs have been classified into two immunologically unrelated groups, the urokinase-type PA (u-PA) (Mr, 55,000) and the tissue-type PA (t-PA) (Mr, 72,000) (3, 4). The latter activator is believed to be the physiological vascular activator (5) and is composed of a single polypeptide chain (6). However, in the presence of plasmin or trypsin it is cleaved at a single site in the central region of the molecule (7), converting it into a two-chain disulfide-linked form. This latter form is composed of a light and a heavy chain, derived from the COOH-terminal and NH2-terminal parts, respectively. The light chain of t-PA contains the active site and is highly homologous to other serine proteases (8). The heavy chain exhibits a number of structural features homologous to structures found in other plasma proteins. Thus, the heavy chain contains two kringle structures (8) similar to those found in prothrombin (9, 10), plasminogen (11), and urokinase (12). It also possesses a growth factor-like domain (12,13) and a domain that shows homology to the fibrin-binding "finger"-like structures of fibronectin (13).Although it has been postulated that exons represent genetic building blocks coding for discrete structural or functional domains (14,15) Analysis of Cloned DNA. The purified DNAs were cleaved with various restriction enzymes either singly or in combination, and the DNA fragments were separated on agarose gels (0.5%-2.0%) or polyacrylamide gels (5%-8%). DNA blotting was carried out according to Southern (22), and the filters were hybridized as described by Moseley et al. (23 5355The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Correlation Of Exon Regions With Structural Units Of Thementioning

confidence: 99%

Section: Correlation Of Exon Regions With Structural Units Of Thementioning

confidence: 99%

The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains.

Ny¹,

Elgh²,

Lund³

1984

Proc. Natl. Acad. Sci. U.S.A.

275

109

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“…When purified from human melanoma cells cultured in the presence of aprotinin, the activator is obtained as a singlechain protein containing a form with 530 residues as well as a proteolytically modified form with 527 residues [6,8,9]. The numbering system for the glycosylation sites discussed in this study (i.e.…”

mentioning

confidence: 99%

Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator

Pohl

Kenne

Nilsson

et al. 1987

European Journal of Biochemistry

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Electrophoretic analysis of endoglycosidase-treated tissue plasminogen activator obtained from human melanoma cells showed that the heterogeneity observed for the protein in these preparations is caused by an Nglycosidically linked N-acetyllactosamine type of carbohydrate chain which is present in about 50% of the molecules. An oligomannose type and an N-acetyllactosamine type of glycan is present in all molecules.Three glycopeptides were isolated and characterized by 'H-NMR, sugar determination, methylation analysis and amino acid determination. The exact attachment site for each of the three glycans could be deduced from the amino acid compositions of the glycopeptides. Asn-117 carries the oligomannose type of glycan, the structure of which was completely determined. Asn-184 is the site where the presence or absence of a biantennary Nacetyllactosamine type of glycan causes the size heterogeneity. The third N-glycosylation site, Asn-448, was found to carry a triantennary or tetraantennary N-acetyllactosamine type of carbohydrate chain.Tissue plasminogen activator, t-PA, is an important regulator of the fibrinolytic activity in the blood [l]. It is released from the vascular endothelium and converts plasminogen to plasmin by a specific proteolytic cleavage. Plasmin in turn efficiently degrades the fibrin network of thrombi formed in the vascular system. The activation of plasminogen by t-PA is stimulated by the presence of fibrin [2, 31, and t-PA binds to fibrin with a high affinity [4]. Thus, plasmin is selectively generated at the site of the fibrin clot which can be lysed without systemic activation of plasminogen in the circulation.The amino acid sequence of the human t-PA molecule has been deduced from isolated cDNA clones [5, 61 and from direct amino acid sequence analysis [7]. Three Asn-Xaa-Ser/ Thr sequences available for N-glycosylation were identified in the structure [7].When purified from human melanoma cells cultured in the presence of aprotinin, the activator is obtained as a singlechain protein containing a form with 530 residues as well as a proteolytically modified form with 527 residues [6,8,9]. The numbering system for the glycosylation sites discussed in this study (i.e. Asn-117, Asn-184 and Asn-448) refers to the shorter form of the single-chain activator in order to follow the nomenclature generally used.If aprotinin is omitted, or if the single-chain t-PA is treated with plasmin, a two-chain form is generated where the Nterminal half (A chain) of the parent molecule is linked to the C-terminal half (B chain) with a disulfide bond. An additional heterogeneity in the melanoma t-PA preparations is an apparent size difference of about 3 kDa [lo]. Analysis of the carbohydrate composition of the separated variants has revealed that the larger molecule is more heavily glycosylated [ 111.The effectiveness of t-PA as a thrombolytic agent has been demonstrated in animal studies [12-141 and recently clinical trials have been initiated with t-PA produced by genetic engineering of animal cells [15, 161...

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“…Thirdly, the single chain activator shows heterogeneity at the NH2-terminal: nearly 50% of the molecules start with an NH2-terminal sequence: GlyAla-Arg-Ser-Tyr-Glu (these are called Lchains), whereas the Gly-Ala-Arg tripeptide is absent in the other form (the S-chain), beginning with Gly/Ser-Tyr-Glu). Finally, certain findings suggest positional heteroge neity (exchange between Gly/Ser) at a single site (position L-4, equivalent to S-l) [19].…”

Section: Physicochemical Properties Of T-pamentioning

confidence: 98%

Tissue-Type Plasminogen Activator

Collen¹,

Lijnen²

1986

Pathophysiol Haemos Thromb

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Differential proteolysis and evidence for a residue exchange in tissue plasminogen activator suggest possible association between two types of protein microheterogeneity

Cited by 50 publications

References 13 publications

The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains.

The structure of the human tissue-type plasminogen activator gene: correlation of intron and exon structures to functional and structural domains.

Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator

Tissue-Type Plasminogen Activator

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