Biomedical subjects

M van Meijer

Publications and source records attributed to M van Meijer.

10 recordsLinked to original sources

PubMed2002

Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease.

Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.

Adenoviruses, Human↗

PubMed1999

A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments.

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.

Animals↗

PubMed1998

Selection of peptides that bind to plasminogen activator inhibitor 1 (PAI-1) using random peptide phage-display libraries.

Large random hexa- and decapenta-peptide libraries were constructed and displayed on the surface of the filamentous phagemid pComb8. Panning of the hexa-peptide library on immobilized plasminogen activator inhibitor 1 (PAI-1) specifically selected a minor fraction of concatemers, indicating that binding to PAI-1 requires an extended amino acid sequence. Accordingly, the decapenta-peptide library exclusively yielded PAI-1 binding peptides of 15 amino acid residues. None of these phage-bound peptides prevented the interaction between PAI-1 and its target serine protease urokinase (u-PA). To isolate peptides that block the interaction between PAI-1 and u-PA, phages bound to immobilized PAI-1 were eluted by incubation with u-PA. Remarkably, this procedure resulted in elution of a unique phage type that harbors a concatemer of decapentamers, consisting of 49 amino acid residues with no obvious similarity to the primary sequence of PAI-1 or u-PA.

Amino Acid Sequence↗

PubMed1997

The suicide substrate reaction between plasminogen activator inhibitor 1 and thrombin is regulated by the cofactors vitronectin and heparin.

The interaction of thrombin with plasminogen activator inhibitor 1 (PAI-1) is shown to result in the simultaneous formation of both cleaved PAI-1 and a sodium dodecyl sulfate-stable thrombin-PAI-1 complex. The kinetics of this reaction can be described by a "suicide substrate" mechanism that includes a branched reaction pathway, which terminates in either the stable inhibitor-enzyme complex or the cleaved inhibitor plus free enzyme. Because of the branched pathway, approximately three moles of PAI-1 are needed to completely inhibit one mole of thrombin. Heparin and vitronectin enhance the rate of inhibition from 9.8 x 10(2) L mol(-1) s(-1) to 6.2 x 10(4) L mol(-1) s(-1) and 2.1 x 10(5) L mol(-1) s(-1), respectively, under optimal conditions. In addition to enhancing the rate of inhibition, both cofactors increase the apparent stoichiometry of the PAI-1-thrombin interaction, with cofactor concentration dependencies similar to the inhibition reaction. Thus, at 37 degrees C approximately six cleavage reactions occur per inhibition reaction. Therefore, thrombin will efficiently inactivate PAI-1 in the presence of either vitronectin or heparin, unless a sufficient excess of the inhibitor is present. These results show that physiological cofactors are able to switch a protease-serpin inhibition reaction to a substrate reaction, depending on the local concentrations of each of the components.

Animals↗

PubMed1997

The composition of complexes between plasminogen activator inhibitor 1, vitronectin and either thrombin or tissue-type plasminogen activator.

Vitronectin (VN) is an obligatory cofactor for the inhibition of thrombin by plasminogen activator inhibitor 1 (PAI-1). It accelerates the rate of association between thrombin and PAI-1 more than two orders of magnitude. In contrast, VN does not accelerate the association between tissue-type plasminogen activator (t-PA) and PAI-1. Previously, we reported that the anti-PAI-1 monoclonal antibody (MoAb) CLB-2C8 binds to a short stretch of amino acids of PAI-1, located between residues 128 and 145, and prevents PAI-1 binding to VN. Furthermore, MoAb CLB-2C8 fully blocks the inhibitory activity of PAI-1 towards t-PA, emphasizing the importance of this area for the interaction with t-PA. Here, we show that this area is also required for the interaction between thrombin and PAI-1, since MoAb CLB-2C8 fully prevents inhibition of thrombin by PAI-1. In spite of similar structural requirements for the interaction between t-PA, PAI-1 and VN and between thrombin, PAI-1 and VN, the intermediate reaction products are clearly distinct. By employing surface plasmon resonance (SPR), using the BIAcore equipment, and by immunoprecipitation we demonstrate that, in the presence of VN, t-PA and PAI-1 form exclusively equimolar binary t-PA/PAI-1 complexes. Thrombin, PAI-1 and VN generate equimolar, binary thrombin/PAI-1 complexes and in addition equimolar, ternary complexes and multimers.

Antibodies, Monoclonal↗

PubMed1996

Selective screening of a large phage display library of plasminogen activator inhibitor 1 mutants to localize interaction sites with either thrombin or the variable region 1 of tissue-type plasminogen activator.

Phage display technology has been exploited to study in detail the interaction between plasminogen activator inhibitor 1 (PAI-1) and either thrombin or an essential positively charged "loop" of tissue-type plasminogen activator (t-PA), denoted variable region 1 (VR1). For this purpose, a PAI-1 mutant phage library was used that served as a reservoir of PAI-1 proteins potentially deficient in the interaction with either VR1 or thrombin. A stringent two-step selection procedure was developed. (i) A negative selection was performed by incubating the pComb3/PAI-1 mutant library with an excess of a thrombin mutant with its VR1 domain substituted with that of t-PA (thrombin-VR1). (ii) The remaining phages were complexed with t-PA (positive selection) and selected by panning with an immobilized anti-t-PA monoclonal antibody. Four consecutive panning rounds yielded an enrichment of pComb3/PAI-1 mutant phages of approximately 50-fold. Sequence analysis of 16 different cDNAs, encoding PAI-1 mutants that are hampered in the binding to thrombin-VR1, revealed the following mutations. Four independent variants share a mutation of the P4' residue (Glu350 --> Lys). Nine independent PAI-1 variants share a substitution of P1' (Met347 --> Lys), whereas three others share a P2 substitution (Ala345 --> Asp). Kinetic analysis of representative PAI-1 mutants provides evidence that the P4' residue is essential for the interaction with the VR1 domain, consistent with the data of Madison et al. (Madison, E.L., Goldsmith, E.J., Gething, M.J., Sambrook, J.F., and Gerard, R.D. (1990) J. Biol. Chem. 265, 21423-21426), whereas the P1' and P2 residues confer thrombin specificity. Concordant with the design of the selection procedure, mutants were obtained that inhibit thrombin-VR1 at least 100-fold slower than wild-type PAI-1, identifying residues that are central to the interaction with either thrombin or VR1. This study demonstrates that phage technology can be used to analyze large numbers of mutants defective in their interaction with other (domains of) proteins, provided an adequate selection scheme is devised.

Alanine↗

PubMed1995

Identification of functional interaction sites on proteins using bacteriophage-displayed random epitope libraries.

We describe a phage-display-based method to identify epitopes or interaction sites on proteins. DNA encoding the protein of interest is partially degraded with DNase I to generate random fragments of 50-200 bp. These fragments are then cloned into a phagemid vector that has been modified to allow the expression of the random fragments and the construction of a (bacterio)phage-displayed random epitope library. Phages displaying functional epitopes can be selected from these libraries by affinity selection or panning. To test this method we have constructed a random-epitope library for human plasminogen-activator inhibitor 1 and used this library to map the epitope of a monoclonal antibody (mAb) directed against this protein. By alignment of the selected overlapping epitope-containing fragments, we were able to locate the epitope of the mAb on a stretch of 39 amino acids spanning from E128 to V166. The approach may also be applied to more complex systems than single-protein genes, such as viral genomes or complete cDNA libraries.

Amino Acid Sequence↗

PubMed1994

Determination of the vitronectin binding site on plasminogen activator inhibitor 1 (PAI-1).

Vitronectin is the carrier protein of plasminogen activator inhibitor 1 (PAI-1). We used a well-characterized panel of anti-human PAI-1 monoclonal antibodies (MoAbs) to localize the vitronectin-binding site on PAI-1. By employing a direct vitronectin/PAI-1 binding assay and two vitronectin-dependent inhibition assays, we demonstrate that the anti-PAI-1 MoAbs CLB-5, CLB-10, CLB-2C8 and I1, directed against different epitopes in the region between amino acids 110 and 145, prevent the interaction of PAI-1 with vitronectin. We conclude that the region between amino acids 110 and 145 of PAI-1 harbours an important determinant for the interaction with vitronectin.

Amino Acid Sequence↗

PubMed1993

Functional display of human plasminogen-activator inhibitor 1 (PAI-1) on phages: novel perspectives for structure-function analysis by error-prone DNA synthesis.

The synthesis of the human plasminogen-activator inhibitor 1 (PAI-1) protein in the cytoplasm of transformed Escherichia coli cells results in inactive protein preparations that can be activated by denaturation and renaturation. We have used the phagemid pComb3, designed for combinatorial immunoglobulin repertoire cloning, for routing of PAI-1 to the periplasm and subsequent exposure on the surface of filamentous phages. Phage-displayed PAI-1 specifically binds to immobilized polyclonal and monoclonal anti-human PAI-1 antibodies. In addition, PAI-1 retains its capacity to form equimolar complexes with its target serine protease tissue-type plasminogen activator (t-PA), as well as its ability to inhibit t-PA activity. Finally, we have explored and manipulated the error-prone property of TaqI DNA polymerase during PCR amplification of the full-length PAI-1 cDNA to generate a large library of predominantly single, random PAI-1 mutants. In addition, a computer simulation program has been devised that converts the number of mutations per codogenic region (in this case PAI-1) into actual mutant proteins. The PAI-1-phage mutant library is composed of 46% single and 34% double mutants and 20% wild-type PAI-1 and can be employed to isolate mutants defective in interactions of PAI-1 with other components. The method described here is applicable to other studies on the structure-function analysis of eukaryotic proteins.

Antibodies, Monoclonal↗

PubMed1992

Cell-associated episialin is a complex containing two proteins derived from a common precursor.

cDNA for the epithelial sialomucin episialin encodes a transmembrane molecule with a large extracellular domain, which mainly consists of repeats of 20 amino acids. Here we confirm the existence of a previously proposed proteolytic cleavage of episialin that occurs in the endoplasmic reticulum (Hilkens, J., and Buijs, F. (1988) J. Biol. Chem. 263, 4215-4222) and show that a similar cleavage takes place in in vitro translation systems. Using in vitro translation of truncated mRNAs, we map the cleavage site to a region located between 71 and 53 amino acids upstream of the transmembrane domain. Analysis of a mutant, in which this region has been deleted, indicates that the cleavage sites used in vitro and in vivo are identical or in close proximity. Both cleavage products remain associated although they are not linked through disulfide bonds. Therefore, the subunit derived from the N terminus, which represents the actual mucin-like domain, remains indirectly anchored to the cell membrane as a result of its interaction with the C-terminal subunit.

Amino Acid Sequence↗