Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1995 Aug 1;182(2):549–557. doi: 10.1084/jem.182.2.549

Mutational analysis of the membrane-proximal cleavage site of L- selectin: relaxed sequence specificity surrounding the cleavage site

PMCID: PMC2192138  PMID: 7543142

Abstract

L-selectin expression is regulated in part by membrane-proximal cleavage from the cell surface of leukocytes and L-selectin-transfected cells. The downregulation of L-selectin from the surface of neutrophils is speculated to be a process involved in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. We previously reported that L-selectin is cleaved between Lys321 and Ser322 in a region that links the second short consensus repeat (SCR) and the transmembrane domain. We demonstrate that replacing this cleavage domain of L-selectin with the corresponding region of E-selectin prevents L-selectin shedding, as judged by inhibiting the generation of the 68-kD soluble and 6-kD transmembrane cleavage products of L- selectin. Unexpectedly, we found that point mutations of the cleavage site, as well as mutations of multiple conserved amino acids within the cleavage domain, do not significantly affect L-selectin shedding. However, short deletions of four or five amino acids in the L-selectin cleavage domain inhibit L-selectin downregulation. Mutations that appeared to inhibit L-selectin shedding resulted in higher levels of cell surface expression, consistent with a lack of apparent proteolysis from the cell membrane. One deletion mutant, I327 delta N332, retains the native cleavage site yet inhibits L-selectin proteolysis as well. Restoring the amino acids deleted between I327 and N332 with five alanine residues restores L-selectin proteolysis. Thus, the proteolytic processing of L-selectin appears to have a relaxed sequence specificity at the cleavage site, and it may depend on the physical length or other secondary structural characteristics of the cleavage domain.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baumhueter S., Dybdal N., Kyle C., Lasky L. A. Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin. Blood. 1994 Oct 15;84(8):2554–2565. [PubMed] [Google Scholar]
  2. Bazil V., Strominger J. L. Metalloprotease and serine protease are involved in cleavage of CD43, CD44, and CD16 from stimulated human granulocytes. Induction of cleavage of L-selectin via CD16. J Immunol. 1994 Feb 1;152(3):1314–1322. [PubMed] [Google Scholar]
  3. Berg E. L., Robinson M. K., Warnock R. A., Butcher E. C. The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor. J Cell Biol. 1991 Jul;114(2):343–349. doi: 10.1083/jcb.114.2.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berg M., James S. P. Human neutrophils release the Leu-8 lymph node homing receptor during cell activation. Blood. 1990 Dec 1;76(11):2381–2388. [PubMed] [Google Scholar]
  5. Bevilacqua M. P., Stengelin S., Gimbrone M. A., Jr, Seed B. Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science. 1989 Mar 3;243(4895):1160–1165. doi: 10.1126/science.2466335. [DOI] [PubMed] [Google Scholar]
  6. Bosenberg M. W., Pandiella A., Massagué J. The cytoplasmic carboxy-terminal amino acid specifies cleavage of membrane TGF alpha into soluble growth factor. Cell. 1992 Dec 24;71(7):1157–1165. doi: 10.1016/s0092-8674(05)80064-9. [DOI] [PubMed] [Google Scholar]
  7. Bowen B. R., Nguyen T., Lasky L. A. Characterization of a human homologue of the murine peripheral lymph node homing receptor. J Cell Biol. 1989 Jul;109(1):421–427. doi: 10.1083/jcb.109.1.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Butcher E. C. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell. 1991 Dec 20;67(6):1033–1036. doi: 10.1016/0092-8674(91)90279-8. [DOI] [PubMed] [Google Scholar]
  9. Butcher E. C. The regulation of lymphocyte traffic. Curr Top Microbiol Immunol. 1986;128:85–122. doi: 10.1007/978-3-642-71272-2_3. [DOI] [PubMed] [Google Scholar]
  10. Dustin M. L., Springer T. A. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature. 1989 Oct 19;341(6243):619–624. doi: 10.1038/341619a0. [DOI] [PubMed] [Google Scholar]
  11. Elwood P. C., Deutsch J. C., Kolhouse J. F. The conversion of the human membrane-associated folate binding protein (folate receptor) to the soluble folate binding protein by a membrane-associated metalloprotease. J Biol Chem. 1991 Feb 5;266(4):2346–2353. [PubMed] [Google Scholar]
  12. Gauss G. H., Lieber M. R. DEAE-dextran enhances electroporation of mammalian cells. Nucleic Acids Res. 1992 Dec 25;20(24):6739–6740. doi: 10.1093/nar/20.24.6739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Green S. A., Setiadi H., McEver R. P., Kelly R. B. The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes. J Cell Biol. 1994 Feb;124(4):435–448. doi: 10.1083/jcb.124.4.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Griffin J. D., Spertini O., Ernst T. J., Belvin M. P., Levine H. B., Kanakura Y., Tedder T. F. Granulocyte-macrophage colony-stimulating factor and other cytokines regulate surface expression of the leukocyte adhesion molecule-1 on human neutrophils, monocytes, and their precursors. J Immunol. 1990 Jul 15;145(2):576–584. [PubMed] [Google Scholar]
  15. Johnston G. I., Cook R. G., McEver R. P. Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell. 1989 Mar 24;56(6):1033–1044. doi: 10.1016/0092-8674(89)90636-3. [DOI] [PubMed] [Google Scholar]
  16. Jutila M. A., Kishimoto T. K., Finken M. Low-dose chymotrypsin treatment inhibits neutrophil migration into sites of inflammation in vivo: effects on Mac-1 and MEL-14 adhesion protein expression and function. Cell Immunol. 1991 Jan;132(1):201–214. doi: 10.1016/0008-8749(91)90019-8. [DOI] [PubMed] [Google Scholar]
  17. Kahn J., Ingraham R. H., Shirley F., Migaki G. I., Kishimoto T. K. Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin. J Cell Biol. 1994 Apr;125(2):461–470. doi: 10.1083/jcb.125.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kishimoto T. K., Jutila M. A., Berg E. L., Butcher E. C. Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. Science. 1989 Sep 15;245(4923):1238–1241. doi: 10.1126/science.2551036. [DOI] [PubMed] [Google Scholar]
  19. Kishimoto T. K., Rothlein R. Integrins, ICAMs, and selectins: role and regulation of adhesion molecules in neutrophil recruitment to inflammatory sites. Adv Pharmacol. 1994;25:117–169. doi: 10.1016/s1054-3589(08)60431-7. [DOI] [PubMed] [Google Scholar]
  20. Kohno T., Brewer M. T., Baker S. L., Schwartz P. E., King M. W., Hale K. K., Squires C. H., Thompson R. C., Vannice J. L. A second tumor necrosis factor receptor gene product can shed a naturally occurring tumor necrosis factor inhibitor. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8331–8335. doi: 10.1073/pnas.87.21.8331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lasky L. A. Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science. 1992 Nov 6;258(5084):964–969. doi: 10.1126/science.1439808. [DOI] [PubMed] [Google Scholar]
  22. Lasky L. A., Singer M. S., Dowbenko D., Imai Y., Henzel W. J., Grimley C., Fennie C., Gillett N., Watson S. R., Rosen S. D. An endothelial ligand for L-selectin is a novel mucin-like molecule. Cell. 1992 Jun 12;69(6):927–938. doi: 10.1016/0092-8674(92)90612-g. [DOI] [PubMed] [Google Scholar]
  23. Lasky L. A., Singer M. S., Yednock T. A., Dowbenko D., Fennie C., Rodriguez H., Nguyen T., Stachel S., Rosen S. D. Cloning of a lymphocyte homing receptor reveals a lectin domain. Cell. 1989 Mar 24;56(6):1045–1055. doi: 10.1016/0092-8674(89)90637-5. [DOI] [PubMed] [Google Scholar]
  24. Levy E., Carman M. D., Fernandez-Madrid I. J., Power M. D., Lieberburg I., van Duinen S. G., Bots G. T., Luyendijk W., Frangione B. Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral hemorrhage, Dutch type. Science. 1990 Jun 1;248(4959):1124–1126. doi: 10.1126/science.2111584. [DOI] [PubMed] [Google Scholar]
  25. McEver R. P., Beckstead J. H., Moore K. L., Marshall-Carlson L., Bainton D. F. GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies. J Clin Invest. 1989 Jul;84(1):92–99. doi: 10.1172/JCI114175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Müllberg J., Schooltink H., Stoyan T., Günther M., Graeve L., Buse G., Mackiewicz A., Heinrich P. C., Rose-John S. The soluble interleukin-6 receptor is generated by shedding. Eur J Immunol. 1993 Feb;23(2):473–480. doi: 10.1002/eji.1830230226. [DOI] [PubMed] [Google Scholar]
  27. Pandiella A., Bosenberg M. W., Huang E. J., Besmer P., Massagué J. Cleavage of membrane-anchored growth factors involves distinct protease activities regulated through common mechanisms. J Biol Chem. 1992 Nov 25;267(33):24028–24033. [PubMed] [Google Scholar]
  28. Pandiella A., Massagué J. Cleavage of the membrane precursor for transforming growth factor alpha is a regulated process. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1726–1730. doi: 10.1073/pnas.88.5.1726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Perez C., Albert I., DeFay K., Zachariades N., Gooding L., Kriegler M. A nonsecretable cell surface mutant of tumor necrosis factor (TNF) kills by cell-to-cell contact. Cell. 1990 Oct 19;63(2):251–258. doi: 10.1016/0092-8674(90)90158-b. [DOI] [PubMed] [Google Scholar]
  30. Porteu F., Nathan C. Shedding of tumor necrosis factor receptors by activated human neutrophils. J Exp Med. 1990 Aug 1;172(2):599–607. doi: 10.1084/jem.172.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ramchandran R., Sen G. C., Misono K., Sen I. Regulated cleavage-secretion of the membrane-bound angiotensin-converting enzyme. J Biol Chem. 1994 Jan 21;269(3):2125–2130. [PubMed] [Google Scholar]
  32. Ravetch J. V., Perussia B. Alternative membrane forms of Fc gamma RIII(CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in single nucleotide substitutions. J Exp Med. 1989 Aug 1;170(2):481–497. doi: 10.1084/jem.170.2.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sahasrabudhe S. R., Spruyt M. A., Muenkel H. A., Blume A. J., Vitek M. P., Jacobsen J. S. Release of amino-terminal fragments from amyloid precursor protein reporter and mutated derivatives in cultured cells. J Biol Chem. 1992 Dec 15;267(35):25602–25608. [PubMed] [Google Scholar]
  34. Schleiffenbaum B., Spertini O., Tedder T. F. Soluble L-selectin is present in human plasma at high levels and retains functional activity. J Cell Biol. 1992 Oct;119(1):229–238. doi: 10.1083/jcb.119.1.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Siegelman M. H., Weissman I. L. Human homologue of mouse lymph node homing receptor: evolutionary conservation at tandem cell interaction domains. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5562–5566. doi: 10.1073/pnas.86.14.5562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Siegelman M. H., van de Rijn M., Weissman I. L. Mouse lymph node homing receptor cDNA clone encodes a glycoprotein revealing tandem interaction domains. Science. 1989 Mar 3;243(4895):1165–1172. doi: 10.1126/science.2646713. [DOI] [PubMed] [Google Scholar]
  37. Sisodia S. S. Beta-amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6075–6079. doi: 10.1073/pnas.89.13.6075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Soubrier F., Alhenc-Gelas F., Hubert C., Allegrini J., John M., Tregear G., Corvol P. Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9386–9390. doi: 10.1073/pnas.85.24.9386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spertini O., Kansas G. S., Munro J. M., Griffin J. D., Tedder T. F. Regulation of leukocyte migration by activation of the leukocyte adhesion molecule-1 (LAM-1) selectin. Nature. 1991 Feb 21;349(6311):691–694. doi: 10.1038/349691a0. [DOI] [PubMed] [Google Scholar]
  40. Springer T. A. Adhesion receptors of the immune system. Nature. 1990 Aug 2;346(6283):425–434. doi: 10.1038/346425a0. [DOI] [PubMed] [Google Scholar]
  41. Subramaniam M., Koedam J. A., Wagner D. D. Divergent fates of P- and E-selectins after their expression on the plasma membrane. Mol Biol Cell. 1993 Aug;4(8):791–801. doi: 10.1091/mbc.4.8.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tedder T. F., Isaacs C. M., Ernst T. J., Demetri G. D., Adler D. A., Disteche C. M. Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-1. Homology with the mouse lymphocyte homing receptor and other human adhesion proteins. J Exp Med. 1989 Jul 1;170(1):123–133. doi: 10.1084/jem.170.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Todd R. F., 3rd, Arnaout M. A., Rosin R. E., Crowley C. A., Peters W. A., Babior B. M. Subcellular localization of the large subunit of Mo1 (Mo1 alpha; formerly gp 110), a surface glycoprotein associated with neutrophil adhesion. J Clin Invest. 1984 Oct;74(4):1280–1290. doi: 10.1172/JCI111538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Watanabe T., Song Y., Hirayama Y., Tamatani T., Kuida K., Miyasaka M. Sequence and expression of a rat cDNA for LECAM-1. Biochim Biophys Acta. 1992 Jul 15;1131(3):321–324. doi: 10.1016/0167-4781(92)90033-v. [DOI] [PubMed] [Google Scholar]
  45. Wong S. T., Winchell L. F., McCune B. K., Earp H. S., Teixidó J., Massagué J., Herman B., Lee D. C. The TGF-alpha precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction. Cell. 1989 Feb 10;56(3):495–506. doi: 10.1016/0092-8674(89)90252-3. [DOI] [PubMed] [Google Scholar]
  46. Yednock T. A., Rosen S. D. Lymphocyte homing. Adv Immunol. 1989;44:313–378. doi: 10.1016/s0065-2776(08)60645-8. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES