Probing for submandibular gland peptide-T receptors on leukocytes with biotinylated-Lys-[Gly](6)-SGP-T.

ABSTRACT

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.