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.

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All data generated or analyzed during this study are included in this article and/or its associated Supplementary Information files. The raw data files are available from the corresponding author upon request. Source data for Figs. 15 and Supplementary Figs. 3, 5, and 10 are available online.

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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

Affiliations

  1. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA

    • Hong Qiu
    • , Songshan Shi
    • , Jingwen Yue
    • , Meng Xin
    • , Alison V. Nairn
    • , Stephanie A. Archer-Hartmann
    • , Mitche Dela Rosa
    • , Melina Galizzi
    • , Shunchun Wang
    • , Parastoo Azadi
    • , Kelley W. Moremen
    •  & Lianchun Wang
  2. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA

    • Hong Qiu
    • , Jingwen Yue
    • , Meng Xin
    • , Kelley W. Moremen
    •  & Lianchun Wang
  3. Institute of Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China

    • Songshan Shi
    •  & Shunchun Wang
  4. Departments of Chemistry and Chemical Biology, Chemical and Biological Engineering, and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA

    • Lei Lin
    • , Xinyue Liu
    • , Fuming Zhang
    •  & Robert J. Linhardt
  5. Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, Ocean University of China, Qingdao, China

    • Guoyun Li
  6. Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, the Netherlands

    • Toin H. van Kuppevelt
  7. Division of Pulmonary and Critical Care Medicine, Department of Medicine, Columbia University Medical Center, New York, NY, USA

    • Wellington V. Cardoso
  8. Multidisciplinary Pain Center, Aichi Medical University, Nagakute, Japan

    • Koji Kimata
  9. Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    • Xingbin Ai
  10. Glycobiology Research and Training Center, Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA

    • Jeffrey D. Esko

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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.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Lianchun Wang.

Integrated supplementary information

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Supplementary Figure 11 PAGE analysis of intact HS extracted from MLEC lines.

    This experiment was carried out one time.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figs. 1–11 and Supplementary Tables 1 and 2

  2. Reporting Summary

  3. Supplementary Data

    Representative profile of the disaccharide composition of HS expressed in the mutant MLECs in HPLC analysis.

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https://doi.org/10.1038/s41592-018-0189-6