Detection of site-specific glycosylation in proteins using flow cytometry.
We tested the possibility that we may express unique peptide probes on cell surfaces, and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications.
Jayakumar
Deepak
D
Chemical and Biological Engineering, State University of New York, Buffalo, New York 14260, USA.
Marathe
Dhananjay D
DD
Neelamegham
Sriram
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001667:Binding, Competitive; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D006031:Glycosylation; D018922:HL-60 Cells; D006639:Histidine / Q000302:isolation & purification; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008863:Microspheres; D008969:Molecular Sequence Data; D009532:Nickel; D009842:Oligopeptides / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013917:Thrombin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; C471213:His-His-His-His-His-His; D008562:Membrane Glycoproteins; D009842:Oligopeptides; C084882:P-selectin ligand protein; D010455:Peptides; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel; D013917:Thrombin