Mutational analysis of the membrane-proximal cleavage site of L-selectin: relaxed sequence specificity surrounding the cleavage site.
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.
Migaki
G I
GI
Department of Immunology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368, USA.
Kahn
J
J
Kishimoto
T K
TK
D000595:Amino Acid Sequence; D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D015536:Down-Regulation; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D019041:L-Selectin; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D016201:Receptors, Lymphocyte Homing / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection
D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D016201:Receptors, Lymphocyte Homing; D019041:L-Selectin; D010450:Endopeptidases