A Transgenic Approach to Live Imaging of Heparan Sulfate Modification Patterns

  • Matthew Attreed
  • Hannes E. BülowEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1229)


Heparan sulfate (HS) glycosaminoglycan chains contain highly modified HS domains that are separated by sections of sparse or no modification. HS domains are central to the role of HS in protein binding and mediating protein–protein interactions in the extracellular matrix. Since HS domains are not genetically encoded, they are impossible to visualize and study with conventional methods in vivo. Here we describe a transgenic approach using previously described single chain variable fragment (scFv) antibodies that bind HS in vitro and on tissue sections with different specificities. By engineering a secretion signal and a fluorescent protein to the scFvs and transgenically expressing these fluorescently tagged antibodies in Caenorhabditis elegans, we are able to directly visualize specific HS domains in live animals (Attreed et al. Nat Methods 9(5):477–479, 2012). The approach allows concomitant colabeling of multiple epitopes, the study of HS dynamics and, could lend itself to a genetic analysis of HS domain biosynthesis or to visualize other nongenetically encoded or posttranslational modifications.

Key words

Heparan sulfate Single chain variable fragment (scFv) antibody, Caenorhabditis elegans Live imaging Nongenetically encoded molecules 



This work was supported by NIH grants F31NS076243 & T32GM07491 (M.A.) and RC1GM090825 & R01GM101313 (H.E.B.).


  1. 1.
    Bernfield M, Götte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, Zako M (1999) Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68:729–777PubMedCrossRefGoogle Scholar
  2. 2.
    Attreed M, Desbois M, van Kuppevelt TH, Bülow HE (2012) Direct visualization of specifically modified extracellular glycans in living animals. Nat Methods 9:477–479PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Fares H, Greenwald I (2001) Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants. Genetics 159:133–145PubMedCentralPubMedGoogle Scholar
  4. 4.
    Esko JD, Selleck SB (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem 71:435–471PubMedCrossRefGoogle Scholar
  5. 5.
    Lindahl U, Kusche-Gullberg M, Kjellen L (1998) Regulated diversity of heparan sulfate. J Biol Chem 273:24979–24982PubMedCrossRefGoogle Scholar
  6. 6.
    Bishop JR, Schuksz M, Esko JD (2007) Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446:1030–1037PubMedCrossRefGoogle Scholar
  7. 7.
    Bülow HE, Hobert O (2006) The molecular diversity of glycosaminoglycans shapes animal development. Annu Rev Cell Dev Biol 22:375–407PubMedCrossRefGoogle Scholar
  8. 8.
    Nadanaka S, Kitagawa H (2008) Heparan sulphate biosynthesis and disease. J Biochem 144:7–14PubMedCrossRefGoogle Scholar
  9. 9.
    Jakobsson L, Kreuger J, Holmborn K, Lundin L, Eriksson I, Kjellen L, Claesson-Welsh L (2006) Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis. Dev Cell 10:625–634PubMedCrossRefGoogle Scholar
  10. 10.
    Sarrazin S, Lamanna WC, Esko JD (2011) Heparan sulfate proteoglycans. Cold Spring Harb Perspect Biol 3Google Scholar
  11. 11.
    Thompson SM, Connell MG, van Kuppevelt TH, Xu R, Turnbull JE, Losty PD, Fernig DG, Jesudason EC (2011) Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation? BMC Dev Biol 11:38PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Hoogenboom HR, Winter G (1992) By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol 227:381–388PubMedCrossRefGoogle Scholar
  13. 13.
    Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222:581–597PubMedCrossRefGoogle Scholar
  14. 14.
    van Kuppevelt TH, Dennissen MA, van Venrooij WJ, Hoet RM, Veerkamp JH (1998) Generation and application of type-specific anti-heparan sulfate antibodies using phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney. J Biol Chem 273:12960–12966PubMedCrossRefGoogle Scholar
  15. 15.
    Dennissen MA, Jenniskens GJ, Pieffers M, Versteeg EM, Petitou M, Veerkamp JH, van Kuppevelt TH (2002) Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies. J Biol Chem 277:10982–10986PubMedCrossRefGoogle Scholar
  16. 16.
    Jenniskens GJ, Oosterhof A, Brandwijk R, Veerkamp JH, van Kuppevelt TH (2000) Heparan sulfate heterogeneity in skeletal muscle basal lamina: demonstration by phage display-derived antibodies. J Neurosci 20:4099–4111PubMedGoogle Scholar
  17. 17.
    Smits NC, Kurup S, Rops AL, ten Dam GB, Massuger LF, Hafmans T, Turnbull JE, Spillmann D, Li JP, Kennel SJ, Wall JS, Shworak NW, Dekhuijzen PN, van der Vlag J, van Kuppevelt TH (2010) The heparan sulfate motif (GlcNS6S-IdoA2S)3, common in heparin, has a strict topography and is involved in cell behavior and disease. J Biol Chem 285:41143–41151PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Smits NC, Lensen JF, Wijnhoven TJ, Ten Dam GB, Jenniskens GJ, van Kuppevelt TH (2006) Phage display-derived human antibodies against specific glycosaminoglycan epitopes. Methods Enzymol 416:61–87PubMedCrossRefGoogle Scholar
  19. 19.
    ten Dam GB, van de Westerlo EM, Smetsers TF, Willemse M, van Muijen GN, Merry CL, Gallagher JT, Kim YS, van Kuppevelt TH (2004) Detection of 2-O-sulfated iduronate and N-acetylglucosamine units in heparan sulfate by an antibody selected against acharan sulfate (IdoA2S-GlcNAc)n. J Biol Chem 279:38346–38352PubMedCrossRefGoogle Scholar
  20. 20.
    van de Westerlo EM, Smetsers TF, Dennissen MA, Linhardt RJ, Veerkamp JH, van Muijen GN, van Kuppevelt TH (2002) Human single chain antibodies against heparin: selection, characterization, and effect on coagulation. Blood 99:2427–2433PubMedCrossRefGoogle Scholar
  21. 21.
    Wijnhoven TJ, Lensen JF, Rops AL, van der Vlag J, Kolset SO, Bangstad HJ, Pfeffer P, van den Hoven MJ, Berden JH, van den Heuvel LP, van Kuppevelt TH (2006) Aberrant heparan sulfate profile in the human diabetic kidney offers new clues for therapeutic glycomimetics. Am J Kidney Dis 48:250–261PubMedCrossRefGoogle Scholar
  22. 22.
    Kurup S, Wijnhoven TJ, Jenniskens GJ, Kimata K, Habuchi H, Li JP, Lindahl U, van Kuppevelt TH, Spillmann D (2007) Characterization of anti-heparan sulfate phage display antibodies AO4B08 and HS4E4. J Biol Chem 282:21032–21042PubMedCrossRefGoogle Scholar
  23. 23.
    Loria PM, Hodgkin J, Hobert O (2004) A conserved postsynaptic transmembrane protein affecting neuromuscular signaling in Caenorhabditis elegans. J Neurosci 24:2191–2201PubMedCrossRefGoogle Scholar
  24. 24.
    Jorgensen EM, Hartwieg E, Schuske K, Nonet ML, Jin Y, Horvitz HR (1995) Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature 378:196–199PubMedCrossRefGoogle Scholar
  25. 25.
    Grant B, Greenwald I (1996) The Caenorhabditis elegans sel-1 gene, a negative regulator of lin-12 and glp-1, encodes a predicted extracellular protein. Genetics 143:237–247PubMedCentralPubMedGoogle Scholar
  26. 26.
    Hermann GJ, Schroeder LK, Hieb CA, Kershner AM, Rabbitts BM, Fonarev P, Grant BD, Priess JR (2005) Genetic analysis of lysosomal trafficking in Caenorhabditis elegans. Mol Biol Cell 16:3273–3288PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94PubMedCentralPubMedGoogle Scholar
  28. 28.
    Evans T (2006) Transformation and microinjection. In: WormBook (ed) The C. elegans research community, WormBook. doi: 10.1895/wormbook.1.108.1,
  29. 29.
    Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88PubMedCrossRefGoogle Scholar
  30. 30.
    Hao L, Johnsen R, Lauter G, Baillie D, Burglin TR (2006) Comprehensive analysis of gene expression patterns of hedgehog-related genes. BMC Genomics 7:280PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Okkema PG, Harrison SW, Plunger V, Aryana A, Fire A (1993) Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans. Genetics 135:385–404PubMedCentralPubMedGoogle Scholar
  32. 32.
    Feil R, Wagner J, Metzger D, Chambon P (1997) Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 237:752–757PubMedCrossRefGoogle Scholar
  33. 33.
    Potter CJ, Tasic B, Russler EV, Liang L, Luo L (2010) The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis. Cell 141:536–548PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Wei X, Potter CJ, Luo L, Shen K (2012) Controlling gene expression with the Q repressible binary expression system in Caenorhabditis elegans. Nat Methods 9:391–395PubMedCrossRefGoogle Scholar
  35. 35.
    Gottschalk A, Schafer WR (2006) Visualization of integral and peripheral cell surface proteins in live Caenorhabditis elegans. J Neurosci Methods 154:68–79PubMedCrossRefGoogle Scholar
  36. 36.
    Filonov GS, Piatkevich KD, Ting LM, Zhang J, Kim K, Verkhusha VV (2011) Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat Biotechnol 29:757–761PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Mello CC, Kramer JM, Stinchcomb D, Ambros V (1991) Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J 10:3959–3970PubMedCentralPubMedGoogle Scholar
  38. 38.
    Frokjaer-Jensen C, Davis MW, Hopkins CE, Newman BJ, Thummel JM, Olesen SP, Grunnet M, Jorgensen EM (2008) Single-copy insertion of transgenes in Caenorhabditis elegans. Nat Genet 40:1375–1383PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Praitis V, Casey E, Collar D, Austin J (2001) Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157:1217–1226PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of GeneticsAlbert Einstein College of Medicine of Yeshiva UniversityBronxUSA
  2. 2.Dominick P. Purpura Department of NeuroscienceAlbert Einstein College of Medicine of Yeshiva UniversityBronxUSA

Personalised recommendations