Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin.

Abstract

Abstract. Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti-cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L-selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys TM and Ser 322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif. EUKOCYTE localization to sites of inflammation is a precisely coordinated multistep process involving distinct families of adhesion receptors and chemokines (9, 56). Regulation of the neutrophil adhesive state is critical for leukocyte function. One major neutrophil adhesion molecule, the Mac-1/3~ integrin (CDIlb/CD18), is both quantitatively upregulated (59) and assumes an active conformation upon neutrophil exposure to chemotactic factors (10). In contrast, another major neutrophil adhesion molecule, L-selectin, is rapidly downregulated upon neutrophil activation (5,17,26,28,29). However, antibodies against either Mac-1 or L-selectin are effective in blocking neutrophil recruitment to sites of inflammation in vivo (28,36,62) and neutrophil adhesion to stimulated endothelial cells in vitro Address all correspondence to Takashi Kei Kishimoto, Ph.D., Boehringer Ingelheim Pharmaceuticals, Department of Immunology 1-5, 900 Ridgebury Road, Ridgefield, CT 06877. (18,20,31,51,5...

Analysis of selective mobilization of L-selectin and Mac-1 reservoirs in bovine neutrophils and eosinophils

“…These data therefore provide evidence for the constitutive shedding of L-selectin from the cell surface in PAF-stimulated granulocytes. Our observations are supported by other reports in which a decreased L-selectin cell surface expression correlated with an increase of extracellular soluble L-selectin [15,17]. In contrast, PAF stimulation could only increase Mac-1 surface expression on eosinophils, whereas neutrophil Mac-1 surface expression remained unaltered.…”

IL-15 Transpresentation Augments CD8+ T Cell Activation and Is Required for Optimal Recall Responses by Central Memory CD8+ T Cells

“…Unfortunately, CD62L is rapidly shed from the cell surface following TCR stimulation through cleavage by the TNF-␣-converting enzyme, thereby making analysis of stimulated populations of bulk or CD44 high CD8 ϩ T cells difficult (45)(46)(47). Therefore, CFSE-labeled CD8 ϩ T cells from LCMV-immune C57BL/6 mice were sorted into naive, T CM , or T EM populations based on CD8␣, CD44, and CD62L expression to Ͼ95% purity (Fig.…”

Effects of selective protein kinase C inhibitors on the proteolytic down-regulation of L-selectin from chemoattractant-activated neutrophils

“…We and others have shown that blocking L-selectin shedding dramatically reduces leukocyte rolling velocities and enhances leukocyte accumulation on endothelial ligands of L-selectin in vitro [13] and in vivo [14], which indicates that L-selectin shedding contributes in regulating L-selectin adhesion. Kahn et al [15] have shown that L-selectin shedding occurs just extracellular to its transmembrane region, resulting in the release of the extracellular portion of L-selectin from the cell surface and retention of a 6-kDa fragment in the cell membrane. Mutational analysis of amino acids at and proximal to the cleavage site of L-selectin revealed that the protease activity is relaxed in sequence specificity but requires certain structural characteristics [16,17].…”