Article | Published:

Remodeling of the lectin–EGF-like domain interface in P- and L-selectin increases adhesiveness and shear resistance under hydrodynamic force

Nature Immunology volume 7, pages 883889 (2006) | Download Citation

Subjects

Abstract

Crystal structures of the lectin and epidermal growth factor (EGF)–like domains of P-selectin show 'bent' and 'extended' conformations. An extended conformation would be 'favored' by forces exerted on a selectin bound at one end to a ligand and at the other end to a cell experiencing hydrodynamic drag forces. To determine whether the extended conformation has higher affinity for ligand, we introduced an N-glycosylation site to 'wedge open' the interface between the lectin and EGF-like domains of P-selectin. This alteration increased the affinity of P-selectin for its ligand P-selectin glycoprotein 1 (PSGL-1) and thereby the strength of P-selectin-mediated rolling adhesion. Similarly, an asparagine-to-glycine substitution in the lectin-EGF-like domain interface of L-selectin enhanced rolling adhesion under shear flow. Our results demonstrate that force, by 'favoring' an extended selectin conformation, can strengthen selectin-ligand bonds.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & The selectins and their ligands. Curr. Opin. Cell Biol. 6, 663–673 (1994).

  2. 2.

    Traffic signals for lymphocyte recirculation and leukocyte emigration: the multi-step paradigm. Cell 76, 301–314 (1994).

  3. 3.

    Selectins and their ligands: Current concepts and controversies. Blood 88, 3259–3287 (1996).

  4. 4.

    & Mechanisms that regulate the function of the selectins and their ligands. Physiol. Rev. 79, 181–213 (1999).

  5. 5.

    & Lymphocyte homing and homeostasis. Science 272, 60–66 (1996).

  6. 6.

    Selectins: lectins that initiate cell adhesion under flow. Curr. Opin. Cell Biol. 14, 581–586 (2002).

  7. 7.

    & Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65, 859–873 (1991).

  8. 8.

    , & Lifetime of the P-selectin: carbohydrate bond and its response to tensile force in hydrodynamic flow. Nature 374, 539–542 (1995).

  9. 9.

    Adhesive interactions of leukocytes, platelets, and the vessel wall during hemostasis and inflammation. Thromb. Haemost. 86, 746–756 (2001).

  10. 10.

    , , & Rolling and transient tethering of leukocytes on antibodies reveal specializations of selectins. Proc. Natl. Acad. Sci. USA 94, 3172–3177 (1997).

  11. 11.

    & An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear. J. Cell Biol. 144, 185–200 (1999).

  12. 12.

    , , , & Dynamic alterations of membrane tethers stabilize leukocyte rolling on P-selectin. Proc. Natl. Acad. Sci. USA 101, 13519–13524 (2004).

  13. 13.

    et al. Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow. J. Cell Biol. 158, 787–799 (2002).

  14. 14.

    , , & Kinetic and mechanical basis of rolling through an integrin and novel Ca2+-dependent rolling and Mg2+-dependent firm adhesion modalities for the α4β7-MAdCAM-1 interaction. Biochemistry 40, 13972–13979 (2001).

  15. 15.

    et al. Direct observation of catch bonds involving cell-adhesion molecules. Nature 423, 190–193 (2003).

  16. 16.

    et al. Low force decelerates L-selectin dissociationfrom P-selectin glycoprotein ligand-1 and endoglycan. J. Biol. Chem. 279, 2291–2298 (2003).

  17. 17.

    et al. Catch bonds govern adhesion through L-selectin at threshold shear. J. Cell Biol. 166, 913–923 (2004).

  18. 18.

    Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu. Rev. Physiol. 57, 827–872 (1995).

  19. 19.

    & Selectins in T-cell recruitment to non-lymphoid tissues and sites of inflammation. Nat. Rev. Immunol. 4, 325–336 (2004).

  20. 20.

    Ligands for L-selectin: homing, inflammation, and beyond. Annu. Rev. Immunol. 22, 129–156 (2004).

  21. 21.

    et al. The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl LewisX oligosaccharide. J. Cell Biol. 117, 895–902 (1992).

  22. 22.

    et al. Expression cloning of a functional glycoprotein ligand for P-selectin. Cell 75, 1179–1186 (1993).

  23. 23.

    , , & Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLeX and PSGL-1. Cell 103, 467–479 (2000).

  24. 24.

    , & Receptor-ligand binding: 'catch' bonds finally caught. Curr. Biol. 13, R611–R613 (2003).

  25. 25.

    , & P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils. J. Cell Biol. 131, 1893–1902 (1995).

  26. 26.

    , , & Molecular mapping of functional domains of the leukocyte receptor for endothelium, LAM-1. J. Cell Biol. 114, 351–358 (1991).

  27. 27.

    , & An activated L-selectin mutant with conserved equilibrium binding properties but enhanced ligand recognition under shear flow. J. Biol. Chem. 275, 18682–18691 (2000).

  28. 28.

    et al. A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion. J. Cell Biol. 124, 609–618 (1994).

  29. 29.

    et al. Insight into E-selectin/ligand interaction from the crystal structure and mutagenesis of the lec/EGF domains. Nature 367, 532–538 (1994).

  30. 30.

    et al. Two-step model of leukocyte-endothelial cell interaction in inflammation: Distinct roles for LECAM-1 and the leukocyte β2 integrins in vivo. Proc. Natl. Acad. Sci. USA 88, 7538–7542 (1991).

  31. 31.

    , & Polymorphism in the human E-selectin gene detected by PCR-SSCP. Hum. Genet. 94, 452–453 (1994).

  32. 32.

    , , , & DNA polymorphisms in adhesion molecule genes–a new risk factor for early atherosclerosis. Hum. Genet. 97, 15–20 (1996).

  33. 33.

    , & Single amino acid residues in the E- and P-selectin epidermal growth factor domains can determine carbohydrate binding specificity. J. Biol. Chem. 271, 16160–16170 (1996).

  34. 34.

    , & Enhanced recruitment of Th2 and CLA-negative lymphocytes by the S128R polymorphism of E-selectin. J. Immunol. 169, 5860–5865 (2002).

  35. 35.

    et al. The S128R polymorphism of E-selectin mediates neuraminidase-resistant tethering of myeloid cells under shear flow. Eur. J. Immunol. 32, 251–260 (2002).

  36. 36.

    et al. Adhesion through L-selectin requires a threshold hydrodynamic shear. Nature 379, 266–269 (1996).

  37. 37.

    , , & Mechanical switching and coupling between two dissociation pathways in a P-selectin adhesion bond. Proc. Natl. Acad. Sci. USA 101, 11281–11286 (2004).

  38. 38.

    et al. Low force decelerates L-selectin dissociation from P-selectin glycoprotein ligand-1 and endoglycan. J. Biol. Chem. 279, 2291–2298 (2004).

  39. 39.

    , , & Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99, 13419–13424 (2002).

  40. 40.

    et al. Extended core 1 and core 2 branched O-glycans differentially modulate Sialyl Lewis x-type L-selectin ligand activity. J. Biol. Chem. 278, 9953–9961 (2003).

  41. 41.

    et al. Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature 343, 757–760 (1990).

  42. 42.

    et al. P- and E- selectin use common sites for carbohydrate ligand recognition and cell adhesion. J. Cell Biol. 120, 1227–1235 (1993).

  43. 43.

    , , , & Preparation of a monoclonal antibody, SZ-51, that recognizes an α-granule membrane protein (GMP-140) on the surface of activated human platelets. Nouv. Rev. Fr. Hematol. 32, 231–235 (1990).

  44. 44.

    et al. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. J. Cell Biol. 128, 661–671 (1995).

  45. 45.

    et al. PADGEM protein: A receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell 59, 305–312 (1989).

  46. 46.

    , , & Interactions of the cytoplasmic domain of P-selectin with clathrin-coated pits enhance leukocyte adhesion under flow. J. Cell Biol. 142, 859–871 (1998).

  47. 47.

    & The α subunit cytoplasmic domain regulates the assembly and adhesiveness of integrin lymphocyte function-associated antigen-1 (LFA-1). J. Immunol. 159, 268–278 (1997).

  48. 48.

    , & C-terminal opening mimics “inside-out” activation of integrin α5β1. Nat. Struct. Biol. 8, 412–416 (2001).

  49. 49.

    , , & One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. Protein Expr. Purif. 23, 440–446 (2001).

  50. 50.

    & Selectin receptor-ligand bonds: Formation limited by shear rate and dissociation governed by the Bell model. Proc. Natl. Acad. Sci. USA 98, 950–955 (2001).

Download references

Acknowledgements

We thank R. Camphausen for suggestions and discussion. Supported by Kirschstein National Research Service Award (F32 AI052851 to U.T.P., F32 GM073529 to T.T.W. and HL-48675 to T.A.S).

Author information

Author notes

    • Uyen T Phan

    Present address: Organon, Cambridge, Massachusetts 02142, USA.

Affiliations

  1. CBR Institute for Biomedical Research, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Uyen T Phan
    • , Travis T Waldron
    •  & Timothy A Springer

Authors

  1. Search for Uyen T Phan in:

  2. Search for Travis T Waldron in:

  3. Search for Timothy A Springer in:

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Timothy A Springer.

Supplementary information

PDF files

  1. 1.

    Supplementary Table 1

    Primers were used in PCR-based mutagenesis to generate the various P-selectin and L-selectin variants.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ni1366

Further reading