Molecular cloning and characterization of a full‐length cDNA clone for human plasminogen

Forsgren, Margaretha; Rådén, Benny; Israelsson, Marianne; Larsson, Kerstin; Hedén, Lars-Olof

doi:10.1016/0014-5793(87)81501-6

Cited by 222 publications

(122 citation statements)

References 21 publications

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“…43 In addition to the Glu460Stop plasminogen mutation, two other homozygous mutations (Arg216→His and Trp597Stop) have recently been identified in two unrelated Turkish girls with ligneous conjunctivitis and severe type I plasminogen deficiency. 17 These molecular genetic findings confirm assumptions by others 4,23,29 that ligneous conjunctivitis is inherited, probably in an autosomal recessive manner.…”

Section: Discussionsupporting

confidence: 72%

“…Single-stranded DNA fragments with a variant migration pattern in single-strand conformation polymorphism analysis were excised from polyacrylamide gel and sequenced as described in the Methods section. The numbering of the nucleotides was carried out according to the method of Forsgren et al, 29 and that of amino acids according to the method of Petersen et al 24 Control…”

Section: Molecular Genetic Findingsmentioning

confidence: 99%

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Therapy with a Purified Plasminogen Concentrate in an Infant with Ligneous Conjunctivitis and Homozygous Plasminogen Deficiency

Schött

Dempfle²,

Beck³

et al. 1998

N Engl J Med

145

114

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Section: Discussionsupporting

confidence: 72%

Section: Molecular Genetic Findingsmentioning

confidence: 99%

Therapy with a Purified Plasminogen Concentrate in an Infant with Ligneous Conjunctivitis and Homozygous Plasminogen Deficiency

Schött

Dempfle²,

Beck³

et al. 1998

N Engl J Med

145

114

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“…Plasmid pREP4 expresses elevated levels of lac repressor, carries the gene for neomycin phosphotransferase and was also purchased from Quiagen. Plasmid pPLGKG [5] contains the complete cDNA sequence of HPg and was kindly provided by Prof. Heden (University of Lund, Sweden).…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

Expression, purification and characterization of the recombinant kringle 2 and kringle 3 domains of human plasminogen and analysis of their binding affinity for ω‐aminocarboxylic acids

Martí

Schaller

Ochensberger

et al. 1994

European Journal of Biochemistry

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The kringle 2 (E161T/C162S/EEE[K2,,,/C169S]TT) and the kringle 3 (TYQ[K3,,]DS) domains of human plasminogen (HPg) were expressed in Eschericlzia coli in an expression vector with the phage T5 promotor/operator element N250PSN250P29 and the cDNA sequence for a hexahistidine tail to facilitate the isolation of the recombinant protein. A coagulation factor Xa (FXa)-sensitive cleavage site was introduced to remove the N-terminal histidine tag. In r-K2, mutations E161T and C162S were introduced to enhance the FXa cleavage yield and C169S to replace the cysteine residue, participating in the inter-kringle disulfide bridge between kringles 2 and 3. Recombinant proteins were isolated by affinity chromatography on Ni"-nitrilotriacetic acid/agarose and refolded under denaturing and reducing conditions followed by a non-denaturing and oxidising environment. The free thiol group in position 297 in r-K3 was selectively alkylated with iodoacetamide. The hexahistidine tail was successfully removed with FXa. The N-terminal sequence, the amino acid composition and the molecular mass analyses are in agreement with the expected data. The correct arrangement of the disulfide bonds was verified by sequence analysis of the corresponding thermolytic and subtilisin fragments. r-K2 exhibits weak binding to lysine-Bio-Gel. The weak binding affinity of r-K2 for o-aminocarboxylic acids is confirmed by intrinsic fluorescence titration with 6-aminohexanoic acid (NH,C,COOH) indicating a Kd of approximately 401 pM. In contrast, r-K3 seems to be devoid of a binding affinity for w-aminocarboxylic acids. Considering earlier determined Kd values of kringle 1, kringle 4 and kringle 5, the binding affinity of HPg kringle domains for NH,C,COOH is proposed to decrease in the following order, kringle 1 > kringle 4 > kringle 5 > kringle 2 > knngle 3.Plasminogen is the main component of the fibrinolytic system and its activated form, plasmin, is responsible for the proteolytic degradation of fibrin in blood clots. Human plasminogen (HPg) is a single-chain proenzyme of 791 amino acids. The sequence has been established by amino acid sequence analysis [l-31 and

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“…Mammalian Pgns of known primary sequence, namely macaca (Tomlinson et al, 1989). porcine (Marti et al, 1985;Schalleret a!., 1987), bovine Berglund et al, 1995), murine (Degen et al,199O), and hedgehog (Lawn et al, 1995: Belczewski et al, 1996, exhibit 65-92% interdomain identity to the human (Forsgren et al, 1987) NTP (Fig. 3).…”

Section: Mm")mentioning

confidence: 99%

Lysine‐50 is a likely site for anchoring the plasminogen N‐terminal peptide to lysine‐binding kringles

Carreño

Martí

et al. 1998

Protein Science

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Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal GlulLys77 peptide (NTP) have been investigated via 'H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant K, < 0.05 mM"). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Ne-acetyl-L-lysine-methyl ester (K, -0.2 mM-'), peptides encompassing Lys50 consistently show higher K, values. Among the investigated linear peptides, N"-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys50-Glu-Gln-NH2 (AcAFYHSKSOEQ-NH,) exhibits the strongest interaction with K4 (KO -1.4 mM"), followed by AcYHSKSOEQ-NH2 (K, -0.9 mM"). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 + Ser mutation was introduced (* AcYHSSSOEQ-NH,), binding was abolished.The Ile27-Ile56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rKI) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5"all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of Pgn (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, K, -4.3 mM" ; rK2, K, -3.7 mM" ; K4, K, -6.4 mM" ; and K5, K, -2.1 mM-' ) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the GIu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.

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Molecular cloning and characterization of a full‐length cDNA clone for human plasminogen

Cited by 222 publications

References 21 publications

Therapy with a Purified Plasminogen Concentrate in an Infant with Ligneous Conjunctivitis and Homozygous Plasminogen Deficiency

Therapy with a Purified Plasminogen Concentrate in an Infant with Ligneous Conjunctivitis and Homozygous Plasminogen Deficiency

Expression, purification and characterization of the recombinant kringle 2 and kringle 3 domains of human plasminogen and analysis of their binding affinity for ω‐aminocarboxylic acids

Lysine‐50 is a likely site for anchoring the plasminogen N‐terminal peptide to lysine‐binding kringles

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