Probing for submandibular gland peptide-T receptors on leukocytes with biotinylated-Lys-[Gly](6)-SGP-T.
Submandibular gland peptide-T (SGP-T) is a potent anti-chemotactic agent for human neutrophils possessing anti-inflammatory properties. Biologically active analogues of SGP-T have been synthesized and a biotinylated form (KG(6)-SGP-T; Bio-KG(6)-SGP-T) was utilized to identify binding sites on isolated human neutrophils. Neutrophils incubated with Bio-KG(6)-SGP-T followed by phycoerythrin (PE)-avidin secondary reagent were fixed and visualized using histochemistry and flow cytometry. At doses of 10(-8) and 10(-9) M, Bio-KG(6)-SGP-T was shown to bind to neutrophils. The binding of Bio-KG(6)-SGP-T, at doses of 10(-8) and 10(-9) M, to neutrophils was abolished by a 100-fold excess of non-biotinylated peptide (KG(6)-SGP-T), but not by 100-fold excess of SGP-T. However, all peptides, dose-dependently reduced the binding of a CD16b antibody (LNK16 clone) to isolated human neutrophils. This discrepancy probably results from different preferred conformations for Bio-KG(6)-SGP-T, KG(6)-SGP-T and SGP-T, since exhaustive conformational searches revealed a high degree of overlap between alpha-Bio-KG(6)-SGP-T and KG(6)-SGP-T that was not seen with SGP-T.
Metwally
Essam
E
Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N-4N1. emetwall@scripps.edu
Befus
A Dean
AD
Davison
Joseph S
JS
Mathison
Ronald
R
D000906:Antibodies; D019921:Biotinylation; D006651:Histocytochemistry; D006801:Humans; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*; D015717:Peptide T / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011487:Protein Conformation; D013363:Submandibular Gland / Q000378:metabolism*
D000906:Antibodies; D010455:Peptides; D015717:Peptide T