A New Method Of Identifying The Expression Of L-selectin On Cells Surface With Oligosaccharide Mimicry Peptides

Purpose Selectin family is one of the adhensive molecular families,there are three members in this family and they are named according to their distribution in vivo :E, P, L.Selectin are Iglycoprotein in the membrane,They have similar structure: N: calcium dependent lectin domain Middle: epidermal growth factor-like domain C:complement regulatory protein.Their gene lies in 1 chromosome.E,P,L-selectin all participate in the congregating and emigrating of the leukocytes to the inflammation site. E,P-selectin mediate the attachment of the tumor cell to endothelium and platelet,indicate they have strong relationship with tumor hematogenous metastasis .L-selectin is well known to facilitate leukocyte rolling along the vessel wall and also required for efficient recirculation of normal lymphocytes into lymph nodes. Fukuda MN isolated the mimicry peptides,IELLQAR,by screening a peptideharboring phage library by using anti-Lewis a antibody.It can bind to L-selectin and inhibit the bound of L-selectin to its ligand competitively in vitro and inhibit inflammation and tumor metastasis to some extent in vivo.Howerer,there is no experimental evidence can prove that it can bind to the L-selectin expressed on cell surface.In our study we first examined the binding activity of monovalent peptides and multivalent peptides to L-selectin expressed on lymphocytes,.make further discussion about the speciality of oligosaccharide mimicry peptides' binding to L-selectin .Meanwhile,we used mimicry peptides to identify the expression of four other tumor cell lines:L1210,P388d1,HCa-F,B16 , compared the identity of binding site of oligosaccharide mimicry peptides and L-selectin antibody ,set up a new method to identify the expression of L-selectin on cells surface with oligosaccharide mimicry peptide.MethodsUse multivalent mimicry peptides ,biotin ,avidin-FITC to bind to L-selectin on cell ,examine the expression of L-selectin,with flow cytometry and fluorescence microscope.Use L-selectin antibody and biotin-multivalent mimicry peptides to bind to L-selectin on cell ,and compare the speciality of the two methods..with laser scanning confocal microscope.Results 1. Flow cytometry.Monovalent mimic peptides can hardly bind with L-selectin while multivalent mimic peptides can bind with L-selectin with high affinity.Compared with multivalent levogyrate mimic peptides, dextrogyrous mimic peptides can also bind to L-selectin with weaker affinity. Soluble L-selectin can effectively inhibit multivalent peptides to bind to lymphocytes,but L-selectin antibody and EDTA didn't have influence on it.2.Fluorescence Microscope:Lymphocyte surface present green fluorescence (excitation at 488nm)3. Laser Scanning Confocal MicroscopeLymphocyte surface present both green and red fluorescence (excitation at 488nm). The superposition of two fluorescence can show the high identity of two methods.Conclusion:1.Multivalent oligosaccharide mimicry peptides can bind to L-selectin on cell surface ,and monovalent mimicry peptides can hardly bind to L-selectin on cell surface2. Multivalent mimicry peptides can bind to L-selectin with high speciality similar to nd L-selectin antibody . .Multivalent oligosaccharide mimicry peptides promise to be a new method to identify the cell surface molecule.