8 results on '"Mertens, Koen"'
Search Results
2. Cellular uptake of coagulation factor VIII: Elusive role of the membrane-binding spikes in the C1 domain.
- Author
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Castro-Núñez L, Koornneef JM, Rondaij MG, Bloem E, van der Zwaan C, Mertens K, Meijer AB, and Meems H
- Subjects
- Cell Line, Humans, Low Density Lipoprotein Receptor-Related Protein-1 metabolism, Models, Molecular, Protein Binding, Protein Domains, Cell Membrane metabolism, Endocytosis, Factor VIII chemistry, Factor VIII metabolism
- Abstract
Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
3. Factor VIII C1 domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake.
- Author
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Bloem E, van den Biggelaar M, Wroblewska A, Voorberg J, Faber JH, Kjalke M, Stennicke HR, Mertens K, and Meijer AB
- Subjects
- Antibodies, Monoclonal metabolism, Antibodies, Monoclonal pharmacology, Binding Sites genetics, Deuterium Exchange Measurement, Factor VIII genetics, Glycosylation, Humans, LDL-Receptor Related Proteins chemistry, LDL-Receptor Related Proteins metabolism, Mass Spectrometry, Models, Molecular, Mutation, Phosphatidylserines chemistry, Phosphatidylserines metabolism, Protein Binding drug effects, Surface Plasmon Resonance, von Willebrand Factor chemistry, von Willebrand Factor metabolism, Endocytosis, Factor VIII chemistry, Factor VIII metabolism, Protein Structure, Tertiary
- Abstract
The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent "hot spot" on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091-2104 and 2157-2162 from hydrogen-deuterium exchange. These comprise the two C1 domain spikes 2092-2093 and 2158-2159. Spike 2092-2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158-2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092-2093 and 2158-2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology.
- Published
- 2013
- Full Text
- View/download PDF
4. Novel role for galectin-8 protein as mediator of coagulation factor V endocytosis by megakaryocytes.
- Author
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Zappelli C, van der Zwaan C, Thijssen-Timmer DC, Mertens K, and Meijer AB
- Subjects
- Cell Differentiation drug effects, Cell Differentiation physiology, Cell Line, Cell Membrane metabolism, Gene Expression Regulation drug effects, Gene Expression Regulation physiology, Humans, Protein Binding physiology, Thrombopoietin pharmacology, Endocytosis physiology, Factor V metabolism, Galectins metabolism, Megakaryocytes metabolism
- Abstract
Galectin-8 (Gal8) interacts with β-galactoside-containing glycoproteins and has recently been implicated to play a role in platelet activation. It has been suggested that Gal8 may also interact with platelet coagulation factor V (FV). This indispensable cofactor is stored in α-granules of platelets via a poorly understood endocytic mechanism that only exists in megakaryocytes (platelet precursor cells). In this study, we now assessed the putative role of Gal8 for FV biology. Surface plasmon resonance analysis and a solid phase binding assay revealed that Gal8 binds FV. The data further show that β-galactosides block the interaction between FV and Gal8. These findings indicate that Gal8 specifically interacts with FV in a carbohydrate-dependent manner. Confocal microscopy studies and flow cytometry analysis demonstrated that megakaryocytic DAMI cells internalize FV. Flow cytometry showed that these cells express Gal8 on their cell surface. Reducing the functional presence of Gal8 on the cells either by an anti-Gal8 antibody or by siRNA technology markedly impaired the endocytic uptake of FV. Compatible with the apparent role of Gal8 for FV uptake, endocytosis of FV was also affected in the presence of β-galactosides. Strikingly, thrombopoietin-differentiated DAMI cells, which represent a more mature megakaryocytic state, not only lose the capacity to express cell-surface bound Gal8 but also lose the ability to internalize FV. Collectively, our data reveal a novel role for the tandem repeat Gal8 in promoting FV endocytosis.
- Published
- 2012
- Full Text
- View/download PDF
5. A single lysine of the two-lysine recognition motif of the D3 domain of receptor-associated protein is sufficient to mediate endocytosis by low-density lipoprotein receptor-related protein.
- Author
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van den Biggelaar M, Sellink E, Klein Gebbinck JW, Mertens K, and Meijer AB
- Subjects
- Amino Acid Motifs, Amino Acid Substitution genetics, Animals, Fluorescein-5-isothiocyanate, Humans, Models, Molecular, Mutant Proteins chemistry, Mutant Proteins metabolism, Protein Binding, Protein Structure, Tertiary, Rats, Structure-Activity Relationship, Surface Plasmon Resonance, Carrier Proteins chemistry, Carrier Proteins metabolism, Endocytosis, LDL-Receptor Related Proteins metabolism, Lysine metabolism
- Abstract
Ligand binding of the low-density lipoprotein (LDL) receptor family is mediated by complement-type repeats (CR) each comprising a binding pocket for a single basic amino acid residue. It has been proposed that at least two CRs are required for high-affinity interaction by utilising two spatially distinct lysine residues on the ligand surface. LDL receptor-related protein (LRP) mediates the cellular uptake of a multitude of ligands, some of which bind LRP with a relatively low affinity suggesting a suboptimal positioning of the two critical lysines. We now addressed the role of the two critical lysines not only in LRP binding but also in LRP-dependent endocytosis. Variants of the third domain (D3) of receptor-associated protein (RAP) were created carrying lysine to alanine or arginine replacements at the putative contact residues K253, K256 and K270. Surface plasmon resonance revealed that replacement of K253 did not affect high-affinity LRP binding at all, whereas replacement of either K256 or K270 markedly reduced the affinity by approximately 10-fold. Binding was abolished when both lysines were replaced. Substitution by either alanine or arginine exerted an almost identical effect on LRP binding. This suggests that despite their positive charge, arginine residues do not support receptor binding at all. Confocal microscopy and flow cytometry studies surprisingly revealed that the single mutants were still taken up and still competed for the uptake of full length RAP despite their receptor binding defect. We therefore propose that the presence of only one of the two critical lysines is sufficient to drive endocytosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
6. Factor VIII interacts with the endocytic receptor low-density lipoprotein receptor-related protein 1 via an extended surface comprising 'hot-spot' lysine residues
- Author
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Van Den Biggelaar, Maartje, Madsen, Jesper J., Faber, Johan H., Zuurveld, Marleen G., Van Der Zwaan, Carmen, Olsen, Ole H., Stennicke, Henning R., Mertens, Koen, Meijer, Alexander B., Sub Pharmaceutical proteins, UIPS - Utrecht Institute for Pharmaceutical Sciences, Sub Pharmaceutical proteins, and UIPS - Utrecht Institute for Pharmaceutical Sciences
- Subjects
Amino Acid Motifs ,Molecular Sequence Data ,Lysine ,Crystallography, X-Ray ,complex mixtures ,Biochemistry ,Mass Spectrometry ,Protein–protein interaction ,Humans ,Amino Acid Sequence ,Receptor ,Molecular Biology ,Binding Sites ,Factor VIII ,Chemistry ,Deuterium Exchange Measurement ,Cell Biology ,Ligand (biochemistry) ,LRP1 ,Endocytosis ,Protein Structure, Tertiary ,Bromodomain ,Lipoproteins, LDL ,Docking (molecular) ,Protein Structure and Folding ,LDL receptor ,bacteria ,lipids (amino acids, peptides, and proteins) ,Low Density Lipoprotein Receptor-Related Protein-1 ,Protein Binding - Abstract
Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule.
- Published
- 2015
7. C1 domain residues Lys 2092 and Phe 2093 are of major importance for the endocytic uptake of coagulation factor VIII
- Author
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Meems, Henriët, van den Biggelaar, Maartje, Rondaij, Mariska, van der Zwaan, Carmen, Mertens, Koen, and Meijer, Alexander B.
- Subjects
- *
ENDOCYTOSIS , *BLOOD coagulation , *METABOLISM , *LOW density lipoproteins , *IMMUNOGLOBULINS , *CELL membranes , *PROTEIN S , *PROTEIN-protein interactions - Abstract
Abstract: Factor VIII (FVIII) catabolism has been demonstrated to involve LDL receptor-related protein (LRP). We have established that antibody fragment KM33 inhibits cofactor function of FVIII by interacting with the membrane binding region 2092–2093 of the C1 domain. As KM33 also inhibits LRP-dependent uptake of FVIII, we now assessed the role of region 2092–2093 for LRP-dependent endocytosis. For this purpose, we employed functional fluorescent FVIII-YFP or -GFP derivatives and U87MG cells which express high levels of LRP. Confocal microscopy studies and flow cytometry analysis combined with siRNA technology showed that the fluorescent FVIII derivatives are indeed effectively internalized by U87MG cells in a LRP-dependent manner. Competition experiments employing an antagonist of the LDL receptor family members revealed that there is a cell surface binding event for FVIII, which is independent of LRP. Cell surface binding proved to be less effective for the FVIII-YFP variants K2092A, F2093A and K2092A/F2093A. Surface plasmon resonance analysis showed that these substitutions affect LRP binding as well. Finally, flow cytometry analysis revealed a major reduction of endocytic uptake of these FVIII-YFP variants. Our results demonstrate that C1 domain residues 2092–2093 are of major importance for FVIII endocytosis by contributing to cell surface binding and receptor binding. [Copyright &y& Elsevier]
- Published
- 2011
- Full Text
- View/download PDF
8. Ins and outs of platelet α-granule proteins
- Author
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Zappelli, C., Pharmaceutics, Mertens, Koen, Meijer, A.B., Mertens, K., and University Utrecht
- Subjects
galectin8 ,STXBP2 ,platelets ,Fibrinogen ,endocytosis ,Integrin ,α-granules ,FactorV ,massspectrometry ,exocytosis - Abstract
Although platelets are small anucleate cell fragments, they are highly organized cells rich in different types of organelles. Platelets contain three secretory granules, dense granules, α-granules and lysosomes which are released upon platelet activation at sites of injury. Most of these α-granule proteins are synthesized by the platelet precursor cells, the megakaryocytes (MKs). Platelets and MKs are, however, unique in that they also take up plasma proteins. The mechanisms behind the endocytosis of plasma proteins as well as the exocytosis machinery are, however, still poorly understood. In our work, we employ protein-protein interaction studies and cell-based internalization studies to get more insight into the mechanisms underlying the endocytosis of plasma proteins in MKs. In addition, we utilize mass spectrometry-based approaches to assess the role of the platelet integrins in α-granule cargo enrichment as well as the mechanism of the release thereof. α-granule coagulation factor V (FV) derives entirely from receptor-mediated endocytosis by megakaryocytes. In our work we provided evidences of a novel role for the lectin galectin8 (Gal8) as mediator of FV uptake. Surface plasmon resonance (SPR) analysis showed a reversible and dose-dependent binding of Gal8 and FV. By inhibiting Gal8 functions and/or targeting its expression in the megakaryocyte-like cells (DAMI), we demonstrated that Gal8-FV complex formation is a prerequisite for FV uptake. Further we explored the potential role of other interaction partners of Gal8, i.e. GPIIbIIIa and GPIb-GPIX-GPV, in FV endocytosis. Mass spectrometry (MS) analysis of platelet lysates from leukocyte adhesion deficiency type III (LAD-III) and Glanzmann's thrombasthenia (GT) patients show that the level of GPV and not of GPIIbIIIa correlates with the amount of FV. Biochemical approaches show that Gal8 can act as a cross-linker between FV and GPV, suggesting a potential role for GPV as Gal8 counter-receptor. Fibrinogen, the most abundant α-granule protein, can be taken up from plasma by both circulating platelets and megakaryocytes. Here, by mass spectrometry analysis of the platelet content from GT and LAD-III patients we explored the role of integrins on platelets cargo enrichment.The results show that GPIIbIIIa only contributes to the uptake of fibrinogen, and that activation of this integrin is not required to facilitate fibrinogen endocytosis. Novel differentially expressed proteins that may be associated with the investigated diseases are identified as well. Moreover MS analysis further revealed the genetic defect of a previously uncharacterized GT patient. In a different line of research, we explored the platelet secretory efficiency of α-granulesin patients with Familial Hemophagocytic Lymphohistiocytosis-5 (FHL-5). Disease-causing mutations have been mapped onto the gene encoding syntaxin-binding-protein2 (Munc18-2). We use MS, flow cytometry and electron microscopy to identify the protein product of a Munc18-2 splice-site variant (1247-1G>C) and the effect thereof on platelet degranulation. This Munc18-2 variant has been established to cause impaired degranulation of neutrophils. In spite of previous suggestions, flow cytometry, electron microscopy and MS studies reveal that the splice-site variant does not significantly affect platelet. Moreover MS analysis revealed the still unknown protein product resulting from the 1247-1G>C mutation.
- Published
- 2014
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