Inflammation results in macrophage activation. This macrophage activation requires participation of B- and T-lymphocytes and a serum factor in the Alpha2-globulin fraction, the vitamin D3 binding protein (designated group-specific component or Gc in human). Our accumulated evidence indicates that the membranous glycosidases of lymphocytes modify the Gc glycoprotein to yield a potent macrophage activating factor. Incubation of Gc protein with a mixture of purified Beta-galactosidase and sialidase efficiently generates the macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Administration of very small amounts (4-10 picograms/ mouse) of in vitro, enzymatically generated macrophage activating factor resulted in greatly enhanced ingestion activity of peritoneal macrophages. In the present application we propose to identify the domain within the Gc protein that is responsible for macrophage activation by three approaches. (1) Fragments of the activated Gc protein will be generated by cyanogen bromide and protease digestion. Peptides will be purified and individually bioassayed for macrophage activation. Particular attention will be given to peptides containing Thr-420, the site of glycosylation. (2) If a small peptide retains macrophage-activating activity, peptides containing the sequence surrounding Thr-420 will be synthesized. N-Acetylgalactosamine will be added chemically and such synthetic peptides will be bioassayed for activity. (3) The macrophage activating domain will be cloned into baculovirus vectors. Large quantities of the glycosylated peptides will be produced in baculovirus-infected insect cells for further study. Our goal is to define a suitable peptide for clinical studies of immunopotentiation.