The Experts below are selected from a list of 141 Experts worldwide ranked by ideXlab platform
Patricia P Wilkins - One of the best experts on this subject based on the ideXlab platform.
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post translational modifications of recombinant p selectin glycoprotein ligand 1 required for binding to p and e selectin
Journal of Biological Chemistry, 1996Co-Authors: Patricia P Wilkins, Suzanne C Crawley, Jasminder WeinsteinAbstract:P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and Tyrosine Sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Galbeta1-3GalNAcalpha1-Se r/Thr); they lack core 2 O-linked glycans (Galbeta1-3(Galbeta1-4GlcNAcbeta1-6)GalNAcalpha1 -Ser/Thr) because they do not express the core 2 beta1 6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack alpha1 3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an alpha1 3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of beta-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be Sulfated. Phenylalanine replacement of three Tyrosines within a consensus sequence for Tyrosine sulfation abolished binding to P-selectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin. PSGL-1 also requires Tyrosine Sulfate to bind P-selectin but not E-selectin.
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Tyrosine sulfation of p selectin glycoprotein ligand 1 is required for high affinity binding to p selectin
Journal of Biological Chemistry, 1995Co-Authors: Patricia P WilkinsAbstract:Abstract P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like glycoprotein on leukocytes that is a high affinity ligand for P-selectin. Previous studies have shown that sialylation and fucosylation of PSGL-1 are required for its binding to P-selectin, but other post-translational modifications of PSGL-1 may also be important. We demonstrate that PSGL-1 synthesized in human HL-60 cells can be metabolically labeled with [S]Sulfate that is incorporated primarily into Tyrosine Sulfate. Treatment of PSGL-1 with a bacterial arylsulfatase releases Sulfate from Tyrosine, resulting in a concordant decrease in binding to P-selectin. These studies demonstrate that Tyrosine Sulfate on PSGL-1 functions in conjunction with sialylated and fucosylated glycans to mediate high affinity binding to P-selectin.
Anders Aspberg - One of the best experts on this subject based on the ideXlab platform.
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the Tyrosine Sulfate domain of fibromodulin binds collagen and enhances fibril formation
Journal of Biological Chemistry, 2016Co-Authors: Viveka Tillgren, Patrik Onnerfjord, Matthias Mörgelin, Sebastian Kalamajski, Anders AspbergAbstract:Small leucine-rich proteoglycans interact with other extracellular matrix proteins and are important regulators of matrix assembly. Fibromodulin has a key role in connective tissues, binding collagen through two identified binding sites in its leucine-rich repeat domain and regulating collagen fibril formation in vitro and in vivo Some nine Tyrosine residues in the fibromodulin N-terminal domain are O-Sulfated, a posttranslational modification often involved in protein interactions. The N-terminal domain mimics heparin, binding proteins with clustered basic amino acid residues. Because heparin affects collagen fibril formation, we investigated whether Tyrosine Sulfate is involved in fibromodulin interactions with collagen. Using full-length fibromodulin and its N-terminal Tyrosine-Sulfated domain purified from tissue, as well as recombinant fibromodulin fragments, we found that the N-terminal domain binds collagen. The Tyrosine-Sulfated domain and the leucine-rich repeat domain both bound to three specific sites along the collagen type I molecule, at the N terminus and at 100 and 220 nm from the N terminus. The N-terminal domain shortened the collagen fibril formation lag phase and Tyrosine sulfation was required for this effect. The isolated leucine-rich repeat domain inhibited the fibril formation rate, and full-length fibromodulin showed a combination of these effects. The fibrils formed in the presence of fibromodulin or its fragments showed more organized structure. Fibromodulin and its Tyrosine Sulfate domain remained bound on the formed fiber. Taken together, this suggests a novel, regulatory function for Tyrosine sulfation in collagen interaction and control of fibril formation. (Less)
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The Tyrosine Sulfate Domain of Fibromodulin Binds Collagen and Enhances Fibril Formation.
The Journal of biological chemistry, 2016Co-Authors: Viveka Tillgren, Matthias Mörgelin, Sebastian Kalamajski, Anders AspbergAbstract:Small leucine-rich proteoglycans interact with other extracellular matrix proteins and are important regulators of matrix assembly. Fibromodulin has a key role in connective tissues, binding collagen through two identified binding sites in its leucine-rich repeat domain and regulating collagen fibril formation in vitro and in vivo Some nine Tyrosine residues in the fibromodulin N-terminal domain are O-Sulfated, a posttranslational modification often involved in protein interactions. The N-terminal domain mimics heparin, binding proteins with clustered basic amino acid residues. Because heparin affects collagen fibril formation, we investigated whether Tyrosine Sulfate is involved in fibromodulin interactions with collagen. Using full-length fibromodulin and its N-terminal Tyrosine-Sulfated domain purified from tissue, as well as recombinant fibromodulin fragments, we found that the N-terminal domain binds collagen. The Tyrosine-Sulfated domain and the leucine-rich repeat domain both bound to three specific sites along the collagen type I molecule, at the N terminus and at 100 and 220 nm from the N terminus. The N-terminal domain shortened the collagen fibril formation lag phase and Tyrosine sulfation was required for this effect. The isolated leucine-rich repeat domain inhibited the fibril formation rate, and full-length fibromodulin showed a combination of these effects. The fibrils formed in the presence of fibromodulin or its fragments showed more organized structure. Fibromodulin and its Tyrosine Sulfate domain remained bound on the formed fiber. Taken together, this suggests a novel, regulatory function for Tyrosine sulfation in collagen interaction and control of fibril formation.
Viveka Tillgren - One of the best experts on this subject based on the ideXlab platform.
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the Tyrosine Sulfate domain of fibromodulin binds collagen and enhances fibril formation
Journal of Biological Chemistry, 2016Co-Authors: Viveka Tillgren, Patrik Onnerfjord, Matthias Mörgelin, Sebastian Kalamajski, Anders AspbergAbstract:Small leucine-rich proteoglycans interact with other extracellular matrix proteins and are important regulators of matrix assembly. Fibromodulin has a key role in connective tissues, binding collagen through two identified binding sites in its leucine-rich repeat domain and regulating collagen fibril formation in vitro and in vivo Some nine Tyrosine residues in the fibromodulin N-terminal domain are O-Sulfated, a posttranslational modification often involved in protein interactions. The N-terminal domain mimics heparin, binding proteins with clustered basic amino acid residues. Because heparin affects collagen fibril formation, we investigated whether Tyrosine Sulfate is involved in fibromodulin interactions with collagen. Using full-length fibromodulin and its N-terminal Tyrosine-Sulfated domain purified from tissue, as well as recombinant fibromodulin fragments, we found that the N-terminal domain binds collagen. The Tyrosine-Sulfated domain and the leucine-rich repeat domain both bound to three specific sites along the collagen type I molecule, at the N terminus and at 100 and 220 nm from the N terminus. The N-terminal domain shortened the collagen fibril formation lag phase and Tyrosine sulfation was required for this effect. The isolated leucine-rich repeat domain inhibited the fibril formation rate, and full-length fibromodulin showed a combination of these effects. The fibrils formed in the presence of fibromodulin or its fragments showed more organized structure. Fibromodulin and its Tyrosine Sulfate domain remained bound on the formed fiber. Taken together, this suggests a novel, regulatory function for Tyrosine sulfation in collagen interaction and control of fibril formation. (Less)
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The Tyrosine Sulfate Domain of Fibromodulin Binds Collagen and Enhances Fibril Formation.
The Journal of biological chemistry, 2016Co-Authors: Viveka Tillgren, Matthias Mörgelin, Sebastian Kalamajski, Anders AspbergAbstract:Small leucine-rich proteoglycans interact with other extracellular matrix proteins and are important regulators of matrix assembly. Fibromodulin has a key role in connective tissues, binding collagen through two identified binding sites in its leucine-rich repeat domain and regulating collagen fibril formation in vitro and in vivo Some nine Tyrosine residues in the fibromodulin N-terminal domain are O-Sulfated, a posttranslational modification often involved in protein interactions. The N-terminal domain mimics heparin, binding proteins with clustered basic amino acid residues. Because heparin affects collagen fibril formation, we investigated whether Tyrosine Sulfate is involved in fibromodulin interactions with collagen. Using full-length fibromodulin and its N-terminal Tyrosine-Sulfated domain purified from tissue, as well as recombinant fibromodulin fragments, we found that the N-terminal domain binds collagen. The Tyrosine-Sulfated domain and the leucine-rich repeat domain both bound to three specific sites along the collagen type I molecule, at the N terminus and at 100 and 220 nm from the N terminus. The N-terminal domain shortened the collagen fibril formation lag phase and Tyrosine sulfation was required for this effect. The isolated leucine-rich repeat domain inhibited the fibril formation rate, and full-length fibromodulin showed a combination of these effects. The fibrils formed in the presence of fibromodulin or its fragments showed more organized structure. Fibromodulin and its Tyrosine Sulfate domain remained bound on the formed fiber. Taken together, this suggests a novel, regulatory function for Tyrosine sulfation in collagen interaction and control of fibril formation.
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the Tyrosine Sulfate rich domains of the lrr proteins fibromodulin and osteoadherin bind motifs of basic clusters in a variety of heparin binding proteins including bioactive factors
Journal of Biological Chemistry, 2009Co-Authors: Viveka Tillgren, Patrik Onnerfjord, Dick HeinegardAbstract:The small leucine-rich repeat proteins, fibromodulin and osteoadherin, have N-terminal extensions with a variable number of O-Sulfated Tyrosine residues. This modification combined with a number of aspartic and glutamic acid residues results in a highly negatively charged domain of less than 30 amino acids. We hypothesized that this domain shares functional properties with heparin regarding binding to proteins and polypeptides containing clusters of basic amino acids. Two other family members, PRELP and chondroadherin, have distinctly different clusters of basic amino acids in their N and C termini, respectively, and PRELP is known to bind to heparin via this domain. Another heparin-binding protein is the cytokine Oncostatin M, with a different cluster of basic amino acids in its C terminus. We used polypeptides representing these basic domains in solid phase assays and demonstrate interactions with the negatively charged N-terminal domain of fibromodulin and full-length osteoadherin. The Tyrosine Sulfate domains also bound heparin-binding proteins such as basic fibroblast growth factor-2, thrombospondin I, MMP13, the NC4 domain of collagen IX, and interleukin-10. Fibronectin with large heparin-binding domains did not bind, neither did CILP containing a heparin-binding thrombospondin type I motif without clustered basic amino acids. Affinity depends on the number and position of the Sulfated Tyrosine residues shown by different binding properties of 10-kDa fragments subfractionated by ion-exchange chromatography. These interactions may sequester growth factors, cytokines, and matrix metalloproteinases in the extracellular matrix as well as contribute to its organization.
Olivier Spertini - One of the best experts on this subject based on the ideXlab platform.
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molecular basis of leukocyte rolling on psgl 1 predominant role of core 2 o glycans and of Tyrosine Sulfate residue 51
Journal of Biological Chemistry, 2003Co-Authors: Michael Pierre Bernimoulin, Xianlu Zeng, Claire Abbal, Sylvain Giraud, Manuel Martinez, Olivier Michielin, Marc Schapira, Olivier SpertiniAbstract:Abstract Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 β1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.
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molecular basis of leukocyte rolling on psgl 1 predominant role of core 2 o glycans and of Tyrosine Sulfate residue 51
Journal of Biological Chemistry, 2003Co-Authors: Michael Pierre Bernimoulin, Xianlu Zeng, Claire Abbal, Sylvain Giraud, Manuel Martinez, Olivier Michielin, Marc Schapira, Olivier SpertiniAbstract:Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.
Fernando Rodriguezpascual - One of the best experts on this subject based on the ideXlab platform.
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differential cleavage of lysyl oxidase by the metalloproteinases bmp1 and adamts2 14 regulates collagen binding through a Tyrosine Sulfate domain
Journal of Biological Chemistry, 2019Co-Authors: Tamara Rosellgarcia, Alain Colige, Laura Dupont, Mourad Bekhouche, Alberto Paradela, Gema Bravo, Fernando RodriguezpascualAbstract:Collagens are the main structural component of the extracellular matrix and provide biomechanical properties to connective tissues. A critical step in collagen fibril formation is the proteolytic removal of N- and C-terminal propeptides from procollagens by metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and BMP1 (bone morphogenetic protein 1)/Tolloid-like families, respectively. BMP1 also cleaves and activates the lysyl oxidase (LOX) precursor, the enzyme catalyzing the initial step in the formation of covalent collagen cross-links, an essential process for fibril stabilization. In this study, using murine skin fibroblasts and HEK293 cells, along with immunoprecipitation, LOX enzymatic activity, solid-phase binding assays, and proteomics analyses, we report that the LOX precursor is proteolytically processed by the procollagen N-proteinases ADAMTS2 and ADAMTS14 between Asp-218 and Tyr-219, 50 amino acids downstream of the BMP1 cleavage site. We noted that the LOX sequence between the BMP1- and ADAMTS-processing sites contains several conserved Tyrosine residues, of which some are post-translationally modified by Tyrosine O-sulfation and contribute to binding to collagen. Taken together, these findings unravel an additional level of regulation in the formation of collagen fibrils. They point to a mechanism that controls the binding of LOX to collagen and is based on differential BMP1- and ADAMTS2/14-mediated cleavage of a Tyrosine-Sulfated domain.
