Abstract
Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure–function relationship of HS has been challenging. Here we report the generation of an HS-mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell. We used this library to (1) determine that the strictly defined fine structure of HS, not its overall degree of sulfation, is more important for FGF2–FGFR1 signaling; (2) define the epitope features of commonly used anti-HS phage display antibodies; and (3) delineate the fine inter-regulation networks by which HS genes modify HS and chain length in mammalian cells at a cell-type-specific level. Our mutant-cell library will allow robust and systematic interrogation of the roles and related structures of HS in a cellular context.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
References
Bernfield, M. et al. Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68, 729–777 (1999).
Esko, J. D. & Lindahl, U. Molecular diversity of heparan sulfate. J. Clin. Invest. 108, 169–173 (2001).
Wang, L., Brown, J. R., Varki, A. & Esko, J. D. Heparin’s anti-inflammatory effects require glucosamine 6-O-sulfation and are mediated by blockade of L- and P-selectins. J. Clin. Invest. 110, 127–136 (2002).
Bishop, J. R., Schuksz, M. & Esko, J. D. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446, 1030–1037 (2007).
Xu, D. & Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu. Rev. Biochem. 83, 129–157 (2014).
Jemth, P. et al. Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs. Exploring the structural diversity by screening for fibroblast growth factor (FGF)1 and FGF2 binding. J. Biol. Chem. 277, 30567–30573 (2002).
Kreuger, J. et al. Fibroblast growth factors share binding sites in heparan sulphate. Biochem. J. 389, 145–150 (2005).
Kamimura, K. et al. Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling. J. Cell Biol. 174, 773–778 (2006).
Lindahl, U. & Li, J. P. Interactions between heparan sulfate and proteins—design and functional implications. Int. Rev. Cell Mol. Biol. 276, 105–159 (2009).
Kraushaar, D. C., Yamaguchi, Y. & Wang, L. Heparan sulfate is required for embryonic stem cells to exit from self-renewal. J. Biol. Chem. 285, 5907–5916 (2010).
Qiu, H. et al. Quantitative phosphoproteomics analysis reveals broad regulatory role of heparan sulfate on endothelial signaling. Mol. Cell. Proteomics 12, 2160–2173 (2013).
Kraushaar, D. C. et al. Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells. J. Biol. Chem. 287, 22691–22700 (2012).
Faham, S., Hileman, R. E., Fromm, J. R., Linhardt, R. J. & Rees, D. C. Heparin structure and interactions with basic fibroblast growth factor. Science 271, 1116–1120 (1996).
Schlessinger, J. et al. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell 6, 743–750 (2000).
Turnbull, J. E., Fernig, D. G., Ke, Y., Wilkinson, M. C. & Gallagher, J. T. Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate. J. Biol. Chem. 267, 10337–10341 (1992).
Ashikari-Hada, S. et al. Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library. J. Biol. Chem. 279, 12346–12354 (2004).
Guimond, S. E. & Turnbull, J. E. Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides. Curr. Biol. 9, 1343–1346 (1999).
Jastrebova, N. et al. Heparan sulfate-related oligosaccharides in ternary complex formation with fibroblast growth factors 1 and 2 and their receptors. J. Biol. Chem. 281, 26884–26892 (2006).
van Kuppevelt, T. H., Dennissen, M. A., van Venrooij, W. J., Hoet, R. M. & Veerkamp, J. H. 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, 12960–12966 (1998).
Dennissen, M. A. et al. Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies. J. Biol. Chem. 277, 10982–10986 (2002).
Thompson, S. M. et al. Heparan sulfate phage display antibodies identify distinct epitopes with complex binding characteristics: insights into protein binding specificities. J. Biol. Chem. 284, 35621–35631 (2009).
Ten Dam, G. B. et al. 3-O-sulfated oligosaccharide structures are recognized by anti-heparan sulfate antibody HS4C3. J. Biol. Chem. 281, 4654–4662 (2006).
Jenniskens, G. J., Oosterhof, A., Brandwijk, R., Veerkamp, J. H. & van Kuppevelt, T. H. Heparan sulfate heterogeneity in skeletal muscle basal lamina: demonstration by phage display-derived antibodies. J. Neurosci. 20, 4099–4111 (2000).
Kurup, S. et al. Characterization of anti-heparan sulfate phage display antibodies AO4B08 and HS4E4. J. Biol. Chem. 282, 21032–21042 (2007).
Deligny, A. et al. NDST2 (N-deacetylase/N-sulfotransferase-2) enzyme regulates heparan sulfate chain length. J. Biol. Chem. 291, 18600–18607 (2016).
Bai, X., Wei, G., Sinha, A. & Esko, J. D. Chinese hamster ovary cell mutants defective in glycosaminoglycan assembly and glucuronosyltransferase I. J. Biol. Chem. 274, 13017–13024 (1999).
Esko, J. D., Rostand, K. S. & Weinke, J. L. Tumor formation dependent on proteoglycan biosynthesis. Science 241, 1092–1096 (1988).
Esko, J. D., Stewart, T. E. & Taylor, W. H. Animal cell mutants defective in glycosaminoglycan biosynthesis. Proc. Natl. Acad. Sci. USA 82, 3197–3201 (1985).
Zhang, L., Lawrence, R., Frazier, B. A. & Esko, J. D. in Methods in Enzymology Vol. 416 (ed Fukuda, M.) 205–221 (Academic Press, New York, 2006).
Xu, X. et al. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat. Biotechnol. 29, 735–741 (2011).
Wijelath, E. et al. Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity. J. Cell. Biochem. 111, 461–468 (2010).
Zhang, B. et al. Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia. J. Clin. Invest. 124, 209–221 (2014).
Qiu, H., Xiao, W., Yue, J. & Wang, L. Heparan sulfate modulates Slit3-induced endothelial cell migration. Methods Mol. Biol. 1229, 549–555 (2015).
Zhang, B. et al. Repulsive axon guidance molecule Slit3 is a novel angiogenic factor. Blood 114, 4300–4309 (2009).
Nakato, H. & Kimata, K. Heparan sulfate fine structure and specificity of proteoglycan functions. Biochim. Biophys. Acta 1573, 312–318 (2002).
Habuchi, H., Habuchi, O. & Kimata, K. Sulfation pattern in glycosaminoglycan: does it have a code? Glycoconj. J. 21, 47–52 (2004).
Maccarana, M., Casu, B. & Lindahl, U. Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor. J. Biol. Chem. 268, 23898–23905 (1993).
Lensen, J. F. et al. Localization and functional characterization of glycosaminoglycan domains in the normal human kidney as revealed by phage display-derived single chain antibodies. J. Am. Soc. Nephrol. 16, 1279–1288 (2005).
Powell, A. K., Yates, E. A., Fernig, D. G. & Turnbull, J. E. Interactions of heparin/heparan sulfate with proteins: appraisal of structural factors and experimental approaches. Glycobiology 14, 17R–30R (2004).
Li, J. P. & Kusche-Gullberg, M. Heparan sulfate: biosynthesis, structure, and function. Int. Rev. Cell Mol. Biol. 325, 215–273 (2016).
Bai, X. & Esko, J. D. An animal cell mutant defective in heparan sulfate hexuronic acid 2-O-sulfation. J. Biol. Chem. 271, 17711–17717 (1996).
Merry, C. L. et al. The molecular phenotype of heparan sulfate in the Hs2st −/− mutant mouse. J. Biol. Chem. 276, 35429–35434 (2001).
Townley, R. A. & Bülow, H. E. Genetic analysis of the heparan modification network in Caenorhabditis elegans. J. Biol. Chem. 286, 16824–16831 (2011).
Inatani, M., Irie, F., Plump, A. S., Tessier-Lavigne, M. & Yamaguchi, Y. Mammalian brain morphogenesis and midline axon guidance require heparan sulfate. Science 302, 1044–1046 (2003).
Wang, L., Fuster, M., Sriramarao, P. & Esko, J. D. Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses. Nat. Immunol. 6, 902–910 (2005).
Forsberg, E. et al. Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme. Nature 400, 773–776 (1999).
Stanford, K. I. et al. Heparan sulfate 2-O-sulfotransferase is required for triglyceride-rich lipoprotein clearance. J. Biol. Chem. 285, 286–294 (2010).
Izvolsky, K. I., Lu, J., Martin, G., Albrecht, K. H. & Cardoso, W. V. Systemic inactivation of Hs6st1 in mice is associated with late postnatal mortality without major defects in organogenesis. Genesis 46, 8–18 (2008).
Sugaya, N., Habuchi, H., Nagai, N., Ashikari-Hada, S. & Kimata, K. 6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture. J. Biol. Chem. 283, 10366–10376 (2008).
Tran, T. H., Shi, X., Zaia, J. & Ai, X. Heparan sulfate 6-O-endosulfatases (Sulfs) coordinate the Wnt signaling pathways to regulate myoblast fusion during skeletal muscle regeneration. J. Biol. Chem. 287, 32651–32664 (2012).
Nagai, N. et al. Involvement of heparan sulfate 6-O-sulfation in the regulation of energy metabolism and the alteration of thyroid hormone levels in male mice. Glycobiology 23, 980–992 (2013).
Vouillot, L., Thélie, A. & Pollet, N. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5, 407–415 (2015).
Liu, W. et al. DSDecode: a web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Mol. Plant 8, 1431–1433 (2015).
Dehairs, J., Talebi, A., Cherifi, Y. & Swinnen, J. V. CRISP-ID: decoding CRISPR mediated indels by Sanger sequencing. Sci. Rep. 6, 28973 (2016).
Nairn, A. V. et al. Glycomics of proteoglycan biosynthesis in murine embryonic stem cell differentiation. J. Proteome Res. 6, 4374–4387 (2007).
Volpi, N. & Linhardt, R. J. High-performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides. Nat. Protoc. 5, 993–1004 (2010).
Skidmore, M. A., Guimond, S. E., Dumax-Vorzet, A. F., Yates, E. A. & Turnbull, J. E. Disaccharide compositional analysis of heparan sulfate and heparin polysaccharides using UV or high-sensitivity fluorescence (BODIPY) detection. Nat. Protoc. 5, 1983–1992 (2010).
Park, Y., Yu, G., Gunay, N. S. & Linhardt, R. J. Purification and characterization of heparan sulphate proteoglycan from bovine brain. Biochem. J. 344, 723–730 (1999).
Chen, Y., Reddy, M., Yu, Y., Zhang, F. & Linhardt, R. J. Glycosaminoglycans from chicken muscular stomach or gizzard. Glycoconj. J. 34, 119–126 (2017).
Acknowledgements
This research was supported by the NIH (R21HL131553, P41GM103390, 5R01HL093339, and U01CA225784 to L.W.; P01HL131474 and P01HL107150 to J.D.E.) and AHA (15POST21260001 and 17SDG33660550 to H.Q). S.S. and S.W. were supported by the Oversea Visiting Scholar Program for Middle-aged and Young Teachers in Shanghai Municipal Universities. We thank K. Howard for English-language revision of the manuscript. We also thank D. Bernsteel and H. Guo in the lab of M. Pierce at the University of Georgia (Athens, GA, USA) for providing the HT-29 cells, and J. Barber and J. Nelson in the flow cytometry core at the University of Georgia for their technical assistance.
Author information
Authors and Affiliations
Contributions
H.Q. and L.W. conceived and designed the research and wrote the manuscript. H.Q. generated all the cell lines. H.Q., S.S., L.L., X.L., G.L., S.A.A.-H., S.W., P.A., F.Z., and R.J.L. designed and performed disaccharide analysis. R.J.L. also contributed to manuscript preparation. H.Q., M.X., and J.Y. performed western blotting. M.D.R., M.G., A.V.N., and K.W.M. performed the transcriptional analysis. T.H.v.K. provided the HS phage display antibodies and contributed to manuscript preparation. K.K., X.A., W.V.C., and J.D.E. provided the transgenic/knockout mice and contributed to manuscript preparation.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Cre-loxP and CRISPR–Cas9 approaches to generate HS-mutant MLEC lines.
The primary MLECs were isolated from mice with HS genes systematically or conditionally targeted (loxP sites flanking the target gene, indicated by “floxed” or “f”). The primary MLECs were immortalized by expression of SV40 T antigen and then single-cell cloned to obtain MLEC lines. Next, the floxed gene was deleted by transient expression of Cre recombinase to derive the ‘daughter’ HS-mutant cell line. In addition, a ‘wild-type’ floxed (Ndst1f/f) MLEC line was cotransfected with target-gene-specific gRNA and Cas9 and then screened for the targeted gene insertion/deletion (indel) mutation and subjected to cell cloning to obtain HS-mutant cell lines. PAM, protospacer-adjacent motif.
Supplementary Figure 2 Expression of endothelial cell markers.
The generated MLEC lines were stained with anti-mouse CD31–FITC or VEGFR2–PE IgG antibody with the corresponding similarly fluorescein-labeled naive IgG staining as background control, and then subjected to flow cytometry analysis. The data shown are representative of 3 independent experiments.
Supplementary Figure 3 The expression patterns of HS biosynthetic and remodeling genes in additional immortalized wild-type MLEC lines.
The gene expression was quantified by qRT-PCR analysis with triplicate repeats. The data are presented as mean ± s.e.m. and are representative of 3 independent experiments.Extended Data. 3
Supplementary Figure 4 Characterization of CRISPR–Cas9-generated HS-mutant MLEC lines.
The Ndst1f/f MLECs were transiently cotransfected with plasmids encoding Cas9 and gRNA targeting Glce, Hs3st1, or Hs3st4 to derive Glce−/− (a), Hs3st1–/ – (b), and Hs3st4−/− (c) cell lines, respectively. The Hs3st1−/− cell line was transiently cotransfected with plasmids expressing Cas9 and gRNA targeting Hs3st4 to derive the Hs3st1−/−;Hs3st4−/− cell line (c). After puromycin selection, the transfected cells were cloned and screened by enzyme mismatch assay for gRNA-induced indel mutation. The identified indel mutations were further characterized by determination of the mutant nucleic acid sequences. Because of induced deletion larger than could be amplified by the PCR primers that amplify the gRNA-targeted region in the wild-type control, no gene sequencing data were obtained for Hs3st4 indels in the Hs3st4−/− and Hs3st1−/−;Hs3st4−/− MLEC lines. Representative results are shown as the mean ± s.e.m. of 3 independent PCR analyses.
Supplementary Figure 5 FGFR1–4 expression in MLEC lines.
(a). qRT-PCR analysis of FGFR1–4 mRNA expression in the wild-type MLEC lines. MLECs express both FGFR1 and FGFR2. Representative data from 3 independent experiments are presented as mean ± s.e.m. (b, c). Western blot analyses of FGFR1 and FGFR2 proteins. In the HS-mutant cell library, all 18 MLEC lines expressed only FGFR1. The human colorectal adenocarcinoma cell line HT29 was included as an FGFR2-expressing positive control. Beta-actin served as a loading control. Representative results from 3 independent western blot experiments are presented, and results of quantitation of the three independent experiments are shown as mean ± s.e.m.Extended Data. 5
Supplementary Figure 6 Whole gel from western blot analysis of FGFR1 and FGFR2 in MLEC lines.
Representative whole gels from three independent western blot analyses of FGFR1 and FGFR2 expression. The original data are presented in Supplementary Fig. 5.
Supplementary Figure 7 Western blot analysis of Erk phosphrylation in the MLEC lines after FGF2 stimulation.
Representative whole gels from three independent western blot analyses of FGFR1 and FGFR2 expression. The original data are presented in Supplementary Fig. 5.
Supplementary Figure 8 Western blot analysis of Erk1/2 phosphorylation in the MLEC lines after FGF2 stimulation.
The MLECs in triplicate were starved in serum-free DMEM for 1 h, stimulated with FGF at 5 ng/ml in serum-free DMEM for 15 min, and then lysed. The resultant cell lysates were analyzed to determine levels of p-Erk1/2 and Erk1/2 in western blots. Representative western blot data of three independent experiments are shown.
Supplementary Figure 9 Whole gels from western blot analysis of Erk phosphorylation in MLECs after FGF2 stimulation.
Whole gels from the western blot analysis of Erk phosphorylation after FGF2 stimulation. The original data are presented in Supplementary Fig. 8.
Supplementary Figure 10 Inter-regulation of HS biosynthesis by HS biosynthetic or remodeling genes.
The qRT-PCR data are representative of 3 independent experiments and are presented as mean ± s.d. P values as listed (two-sided, unpaired Student’s t-test).Extended Data. 10
Supplementary Figure 11 PAGE analysis of intact HS extracted from MLEC lines.
This experiment was carried out one time.
Supplementary information
Supplementary Text and Figures
Supplementary Figs. 1–11 and Supplementary Tables 1 and 2
Supplementary Data
Representative profile of the disaccharide composition of HS expressed in the mutant MLECs in HPLC analysis.
Rights and permissions
About this article
Cite this article
Qiu, H., Shi, S., Yue, J. et al. A mutant-cell library for systematic analysis of heparan sulfate structure–function relationships. Nat Methods 15, 889–899 (2018). https://doi.org/10.1038/s41592-018-0189-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41592-018-0189-6
This article is cited by
-
Molecular mechanism of decision-making in glycosaminoglycan biosynthesis
Nature Communications (2023)
-
Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis
Scientific Reports (2022)
-
Chemical editing of proteoglycan architecture
Nature Chemical Biology (2022)
-
Structural characteristics of Heparan sulfate required for the binding with the virus processing Enzyme Furin
Glycoconjugate Journal (2022)
-
Glycosaminoglycan Domain Mapping of Cellular Chondroitin/Dermatan Sulfates
Scientific Reports (2020)