Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator

Li, X.-K; Lijnen, H. Roger; Nelles, Luc; Hu, Min; Collen, D.

doi:10.1016/0167-4838(92)90072-l

Cited by 10 publications

(3 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the present study, the overexpressed mutant uPA protein, which had glutamine as the 302nd amino acid residue instead of asparagine and was deficient in N-glycans, altered the invasiveness of liver cancer cells. Therefore, the glycosylation site at asparagine in the 302nd amino acid residue of uPA was found to be an important factor in controlling cancer cell invasiveness, although the glycosylation pattern of uPA does not affect its interaction with plasma proteins directly involved in its fibrinolytic function (32); additionally, the fibrinolytic activity of recombinant non-glycosylated single-chain uPA has been demonstrated to be similar to that of the glycosylated urinary and recombinant proteins (33).…”

Section: Discussionmentioning

confidence: 99%

Analysis of the correlation between alterations in N‑glycans and invasiveness in liver cancer cell lines

et al. 2020

View full text Add to dashboard Cite

The N-glycoforms of glycoproteins modify protein function and control a number of biological pathways. The aim of the present study was to investigate the correlation between alterations in N-glycans and cancer aggressiveness in terms of cancer cell invasion ability. The expression of urokinase-type plasminogen activator (uPA) and N-acetylglucosaminyltr ansferase V (GnT-V) in liver cancer cell lines was analyzed by western blotting. Cell invasiveness was analyzed by Matrigel invasion assays. uPA and GnT-V expression in liver cancer cell lines was knocked down by RNA interference. Furthermore, uPA was overexpressed in liver cancer cells using lentiviral vectors, and a mutant strain of HepG2 cells overexpressing uPA deficient in N-glycans was established. A glycoblotting-assisted matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry-based quantitative analysis of liver cancer cell lines was performed, in which invasiveness was altered by modifying the expression of uPA and GnT-V. N-glycan profiles were found to differ between the highly invasive liver cancer cell line HLE and the less invasive cell line HepG2. The expression of several N-glycans, including a form with m/z=1892, was changed according to invasiveness controlled by knockdown and overexpression of uPA. The invasiveness of HepG2 cells with mutant uPA did not increase regardless of the level of expression of uPA. Following GnT-V knockdown and N-glycan alteration, uPA expression did not change, whereas cell invasiveness decreased. One N-glycan (m/z=1892) was common among N-glycans in the comparative analysis between HLE and HepG2, HLE and uPA knockdown HLE, HepG2 and uPA-overexpressing HepG2, and HLE and GnT-V knockdown HLE cells and among N-glycan profiles in human uPA. Therefore, N-glycosylation is an important factor controlling invasiveness of liver cancer cells, and a specific N-glycan (m/z=1892) associated with the invasion of liver cancer cells via uPA was identified in the present study.

show abstract

Section: Discussionmentioning

confidence: 99%

Analysis of the correlation between alterations in N‑glycans and invasiveness in liver cancer cell lines

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Thus, deletion of residues Asn 2 through Phe IS7 did not affect the rapid in vivo clearance of rscu-PA, but resulted in abolition of the enzymatic activity (3S). Furthermore, the fibrinolytic activity and the in vivo turnover of rscu-PA were not affected by the absence of carbohydrate as a result of site-specific mutagenesis of the unique glycosylated Asn 302 residue to GIn (36).…”

Section: Rscu-pa-32kmentioning

confidence: 98%

Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

et al. 1994

Self Cite

View full text Add to dashboard Cite

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

show abstract

“…This carbohydrate moiety is known to contribute to the active site conformation as previous studies have shown that its absence affects uPA's catalytic properties (Sarubbi et al 1989;Lenich et al 1992;Li et al 1992). …”

Section: Glycosylation Analysismentioning

confidence: 99%

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

et al. 2005

View full text Add to dashboard Cite

The development of specific catalytic inhibitors for the serine protease urokinase-type plasminogen activator (uPA) has been hindered due to difficulties in producing sufficient amounts of active recombinant uPA that is catalytically equivalent to native uPA. The purpose of this study was to develop an efficient system for the expression of recombinant human uPA that exhibits comparable proteolytic activity to that of the native protein. Since post-translational modifications (e.g. glycosylations) of uPA are necessary for efficient proteolytic activity, we have used a mammalian cell line [Chinese hamster ovary (CHO)-S] to express recombinant human uPA. CHO-S cells were selected to stably express full-length recombinant human uPA containing a hexahistidine tag at its C-terminus to permit purification by nickel-based affinity chromatography. Secretion of recombinant uPA into the culture media was confirmed by immunoblotting and the presence of an N-linked glycosylation was confirmed by PNGase sensitivity. Enzymatic activity of purified recombinant uPA was demonstrated using zymography and quantitatively compared to native uPA by kinetic analysis using an uPA-specific substrate. Native uPA and the recombinant uPA demonstrated comparable K m values (55.7 and 39 lM, respectively). Furthermore, inhibition studies using benzamidine resulted in a K i of 195 lM for native uPA, while recombinant uPA had a K i of 112 lM. These data indicate that recombinant human uPA expressed by CHO-S cells is functionally comparable to native uPA.Abbreviations: CHO-S -Chinese hamster ovary

show abstract

Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator

Cited by 10 publications

References 32 publications

Analysis of the correlation between alterations in N‑glycans and invasiveness in liver cancer cell lines

Analysis of the correlation between alterations in N‑glycans and invasiveness in liver cancer cell lines

Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

Contact Info

Product

Resources

About