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Home / Equine Research Bank / Biochem J / Topological assignment of the N-terminal extension of plasma...
The Biochemical journal2004; 385(Pt 3); 659-665; doi: 10.1042/BJ20040875

Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface.

Abstract: The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34-Ile45 and Leu64-Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.
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Publication Date: 2004-09-21 PubMed ID: 15377282PubMed Central: PMC1134740DOI: 10.1042/BJ20040875Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research focuses on studying more about the actin-binding protein gelsolin, particularly, the vertebrates’ plasma gelsolin by identifying its N-terminal sequence’s positioning. The whole process was found out through a biochemical and immunological approach, and a model of it was created.

Understanding The Research

  • The research starts by talking about gelsolin, a protein that binds with actin. Scattered widely in the animal kingdom, this protein comes in two versions, a cytoplasmic one and an extracellular one.
  • The difference between the two is that the plasma gelsolin – an extracellular variant, comes with an N-terminal extension. This feature makes it secreted by many cell types, thus equally present in cells and tissues with cytoplasmic gelsolin.
  • While the tertiary structure of plasma gelsolin has been exposed, information about the N-terminal extension was missing.

Methodological Approach

  • To comprehend the structure of the N-terminal extension, the study utilizes methods that include biochemical and immunological approaches.
  • A special monoclonal antibody is formulated for this purpose that can only recognize the cytoplasmic gelsolin but not the extracellular one. The antibody thus enables the study to immunodetect and quantify cytoplasmic gelsolin in the company of plasma gelsolin.

Findings

  • Through limited proteolysis and pepscan analysis, researchers were able to map the binding epitope and locate it within two regions in the cytoplasmic gelsolin sequence: Tyr34-Ile45 and Leu64-Ile78.
  • In the cytoplasmic variant’s tertiary structure, these sequences are mutually adjacent and lie near the N-terminus.
  • The inference is that the antibody’s binding site gets covered by the N-terminal extension in plasma gelsolin, thus preventing antibody binding.
  • The research thus manages to ascertain a topological model of the N-terminal extension on the gelsolin molecule’s surface, which wasn’t known earlier.

Cite This Article

APA
Fock U, Jockusch BM, Schubert WD, Hinssen H. (2004). Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface. Biochem J, 385(Pt 3), 659-665. https://doi.org/10.1042/BJ20040875

Publication

The Biochemical journal
ISSN: 1470-8728
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 385
Issue: Pt 3
Pages: 659-665

Researcher Affiliations

Fock, Ulrike
  • Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38106 Braunschweig, Germany.
Jockusch, Brigitte M
    Schubert, Wolf-Dieter
      Hinssen, Horst

        MeSH Terms

        • Amino Acid Sequence
        • Animals
        • Antibodies, Monoclonal / immunology
        • Antibody Specificity
        • Binding Sites
        • Cytoplasm / metabolism
        • Enzyme-Linked Immunosorbent Assay
        • Epitope Mapping
        • Epitopes / chemistry
        • Epitopes / immunology
        • Epitopes / metabolism
        • Gelsolin / chemistry
        • Gelsolin / genetics
        • Gelsolin / immunology
        • Gelsolin / metabolism
        • Genetic Variation
        • Humans
        • Models, Molecular
        • Molecular Sequence Data
        • Protein Conformation
        • Sensitivity and Specificity
        • Swine

        References

        This article includes 37 references
        1. Burtnick LD, Robinson RC, Choe S. Structure and function of gelsolin.. Results Probl Cell Differ 2001;32:201-11.
          pubmed: 11131832doi: 10.1007/978-3-540-46560-7_14google scholar: lookup
        2. Sun HQ, Yamamoto M, Mejillano M, Yin HL. Gelsolin, a multifunctional actin regulatory protein.. J Biol Chem 1999 Nov 19;274(47):33179-82.
          pubmed: 10559185doi: 10.1074/jbc.274.47.33179google scholar: lookup
        3. McGough AM, Staiger CJ, Min JK, Simonetti KD. The gelsolin family of actin regulatory proteins: modular structures, versatile functions.. FEBS Lett 2003 Sep 25;552(2-3):75-81.
          pubmed: 14527663doi: 10.1016/s0014-5793(03)00932-3google scholar: lookup
        4. Kwiatkowski DJ. Functions of gelsolin: motility, signaling, apoptosis, cancer.. Curr Opin Cell Biol 1999 Feb;11(1):103-8.
          pubmed: 10047530doi: 10.1016/s0955-0674(99)80012-xgoogle scholar: lookup
        5. Hellweg T, Hinssen H, Eimer W. The Ca(2+)-induced conformational change of gelsolin is located in the carboxyl-terminal half of the molecule.. Biophys J 1993 Aug;65(2):799-805.
          pmc: PMC1225780pubmed: 8218904doi: 10.1016/s0006-3495(93)81121-4google scholar: lookup
        6. Robinson RC, Mejillano M, Le VP, Burtnick LD, Yin HL, Choe S. Domain movement in gelsolin: a calcium-activated switch.. Science 1999 Dec 3;286(5446):1939-42.
          pubmed: 10583954doi: 10.1126/science.286.5446.1939google scholar: lookup
        7. Lin KM, Mejillano M, Yin HL. Ca2+ regulation of gelsolin by its C-terminal tail.. J Biol Chem 2000 Sep 8;275(36):27746-52.
          pubmed: 10862770doi: 10.1074/jbc.m003732200google scholar: lookup
        8. Kolappan S, Gooch JT, Weeds AG, McLaughlin PJ. Gelsolin domains 4-6 in active, actin-free conformation identifies sites of regulatory calcium ions.. J Mol Biol 2003 May 23;329(1):85-92.
          pubmed: 12742020doi: 10.1016/s0022-2836(03)00383-8google scholar: lookup
        9. Choe H, Burtnick LD, Mejillano M, Yin HL, Robinson RC, Choe S. The calcium activation of gelsolin: insights from the 3A structure of the G4-G6/actin complex.. J Mol Biol 2002 Dec 6;324(4):691-702.
          pubmed: 12460571doi: 10.1016/s0022-2836(02)01131-2google scholar: lookup
        10. Narayan K, Chumnarnsilpa S, Choe H, Irobi E, Urosev D, Lindberg U, Schutt CE, Burtnick LD, Robinson RC. Activation in isolation: exposure of the actin-binding site in the C-terminal half of gelsolin does not require actin.. FEBS Lett 2003 Sep 25;552(2-3):82-5.
          pubmed: 14527664doi: 10.1016/s0014-5793(03)00933-5google scholar: lookup
        11. Bryan J. Gelsolin has three actin-binding sites.. J Cell Biol 1988 May;106(5):1553-62.
          pmc: PMC2115046pubmed: 2836434doi: 10.1083/jcb.106.5.1553google scholar: lookup
        12. Khaitlina S, Hinssen H. Ca-dependent binding of actin to gelsolin.. FEBS Lett 2002 Jun 19;521(1-3):14-8.
          pubmed: 12067717doi: 10.1016/s0014-5793(02)02657-1google scholar: lookup
        13. Kwiatkowski DJ, Stossel TP, Orkin SH, Mole JE, Colten HR, Yin HL. Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain.. Nature 1986 Oct 2-8;323(6087):455-8.
          pubmed: 3020431doi: 10.1038/323455a0google scholar: lookup
        14. Kwiatkowski DJ, Mehl R, Izumo S, Nadal-Ginard B, Yin HL. Muscle is the major source of plasma gelsolin.. J Biol Chem 1988 Jun 15;263(17):8239-43.
          pubmed: 2836420
        15. Way M, Weeds A. Nucleotide sequence of pig plasma gelsolin. Comparison of protein sequence with human gelsolin and other actin-severing proteins shows strong homologies and evidence for large internal repeats.. J Mol Biol 1988 Oct 20;203(4):1127-33.
          pubmed: 2850369doi: 10.1016/0022-2836(88)90132-5google scholar: lookup
        16. Burtnick LD, Koepf EK, Grimes J, Jones EY, Stuart DI, McLaughlin PJ, Robinson RC. The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation.. Cell 1997 Aug 22;90(4):661-70.
          pubmed: 9288746doi: 10.1016/s0092-8674(00)80527-9google scholar: lookup
        17. Hinssen H, Small JV, Sobieszek A. A Ca2+-dependent actin modulator from vertebrate smooth muscle.. FEBS Lett 1984 Jan 23;166(1):90-5.
          pubmed: 6537923doi: 10.1016/0014-5793(84)80051-4google scholar: lookup
        18. Pope B, Gooch J, Hinssen H, Weeds AG. Loss of calcium sensitivity of plasma gelsolin is associated with the presence of calcium ions during preparation.. FEBS Lett 1989 Dec 18;259(1):185-8.
          pubmed: 2557242doi: 10.1016/0014-5793(89)81524-8google scholar: lookup
        19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature 1970 Aug 15;227(5259):680-5.
          pubmed: 5432063doi: 10.1038/227680a0google scholar: lookup
        20. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose.. J Biochem Biophys Methods 1984 Dec;10(3-4):203-9.
          pubmed: 6530509doi: 10.1016/0165-022x(84)90040-xgoogle scholar: lookup
        21. Hinssen H, Vandekerckhove J, Lazarides E. Gelsolin is expressed in early erythroid progenitor cells and negatively regulated during erythropoiesis.. J Cell Biol 1987 Sep;105(3):1425-33.
          pmc: PMC2114820pubmed: 2821013doi: 10.1083/jcb.105.3.1425google scholar: lookup
        22. Frank R. Spot-synthesis. An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support.. Tetrahedron 1992;48:9217–9232.
        23. Mayboroda O, Schlüter K, Jockusch BM. Differential colocalization of profilin with microfilaments in PtK2 cells.. Cell Motil Cytoskeleton 1997;37(2):166-77.
          pubmed: 9186014doi: 10.1002/(sici)1097-0169(1997)37:2<166::aid-cm9>3.0.co;2-6google scholar: lookup
        24. Gonsior SM, Platz S, Buchmeier S, Scheer U, Jockusch BM, Hinssen H. Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody.. J Cell Sci 1999 Mar;112 ( Pt 6):797-809.
          pubmed: 10036230doi: 10.1242/jcs.112.6.797google scholar: lookup
        25. Koepf EK, Hewitt J, Vo H, Macgillivray RT, Burtnick LD. Equus caballus gelsolin--cDNA sequence and protein structural implications.. Eur J Biochem 1998 Feb 1;251(3):613-21.
          pubmed: 9490033doi: 10.1046/j.1432-1327.1998.2510613.xgoogle scholar: lookup
        26. Nodes BR, Shackelford JE, Lebherz HG. Synthesis and secretion of serum gelsolin by smooth muscle tissue.. J Biol Chem 1987 Apr 15;262(11):5422-7.
          pubmed: 3031054
        27. Chaponnier C, Janmey PA, Yin HL. The actin filament-severing domain of plasma gelsolin.. J Cell Biol 1986 Oct;103(4):1473-81.
          pmc: PMC2114347pubmed: 3021782doi: 10.1083/jcb.103.4.1473google scholar: lookup
        28. McLaughlin PJ, Gooch JT, Mannherz HG, Weeds AG. Structure of gelsolin segment 1-actin complex and the mechanism of filament severing.. Nature 1993 Aug 19;364(6439):685-92.
          pubmed: 8395021doi: 10.1038/364685a0google scholar: lookup
        29. Irobi E, Burtnick LD, Urosev D, Narayan K, Robinson RC. From the first to the second domain of gelsolin: a common path on the surface of actin?. FEBS Lett 2003 Sep 25;552(2-3):86-90.
          pubmed: 14527665doi: 10.1016/s0014-5793(03)00934-7google scholar: lookup
        30. Jones TA, Zou JY, Cowan SW, Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models.. Acta Crystallogr A 1991 Mar 1;47 ( Pt 2):110-9.
          pubmed: 2025413doi: 10.1107/s0108767390010224google scholar: lookup
        31. Hiyoshi M, Nakao Y, Tatsumi N. Comparison of two monoclonal antibodies, H6B11 and GS2C4, against human plasma gelsolin.. Biochem Mol Biol Int 1994 Mar;32(4):755-62.
          pubmed: 8038725
        32. Teubner A, Sobek-Klocke I, Hinssen H, Eichenlaub-Ritter U. Distribution of gelsolin in mouse ovary.. Cell Tissue Res 1994 Jun;276(3):535-44.
          pubmed: 8062342doi: 10.1007/bf00343950google scholar: lookup
        33. Dissmann E, Hinssen H. Immunocytochemical localization of gelsolin in fibroblasts, myogenic cells, and isolated myofibrils.. Eur J Cell Biol 1994 Apr;63(2):336-44.
          pubmed: 8082657
        34. Carron CP, Hwo SY, Dingus J, Benson DM, Meza I, Bryan J. A re-evaluation of cytoplasmic gelsolin localization.. J Cell Biol 1986 Jan;102(1):237-45.
          pmc: PMC2114051pubmed: 3001100doi: 10.1083/jcb.102.1.237google scholar: lookup
        35. Allen PG. Functional consequences of disulfide bond formation in gelsolin.. FEBS Lett 1997 Jan 13;401(1):89-94.
          pubmed: 9003812doi: 10.1016/s0014-5793(96)01439-1google scholar: lookup
        36. Wen D, Corina K, Chow EP, Miller S, Janmey PA, Pepinsky RB. The plasma and cytoplasmic forms of human gelsolin differ in disulfide structure.. Biochemistry 1996 Jul 30;35(30):9700-9.
          pubmed: 8703941doi: 10.1021/bi960920ngoogle scholar: lookup
        37. Dieffenbach CW, SenGupta DN, Krause D, Sawzak D, Silverman RH. Cloning of murine gelsolin and its regulation during differentiation of embryonal carcinoma cells.. J Biol Chem 1989 Aug 5;264(22):13281-8.
          pubmed: 2546951

        Citations

        This article has been cited 1 times.
        1. Piktel E, Levental I, Durnaś B, Janmey PA, Bucki R. Plasma Gelsolin: Indicator of Inflammation and Its Potential as a Diagnostic Tool and Therapeutic Target.. Int J Mol Sci 2018 Aug 25;19(9).
          doi: 10.3390/ijms19092516pubmed: 30149613google scholar: lookup
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