alpha-chymotrypsin and galactocerebroside

There is increasing evidence that the ligation of adhesion molecules such as L-selectin can activate phagocytes to their full inflammatory potential. Sulfatide has been established as ligand for L-selectin and shown to trigger intracellular signals in human neutrophils. However, it remains unclear whether the ligation of L-selectin with sulfatide affects neutrophil phagocytosis. We studied the effects of sulfatide upon Fc gamma R- and CR3-mediated human neutrophil phagocytosis. Adhesion of the cells to a sulfatide-coated surface resulted in a dose-dependent enhancement of phagocytosis mediated via Fc gamma R or CR3, or both receptors. Galactocerebroside, but not glucocerebroside, also enhanced phagocytosis by neutrophils; therefore, galactose residue is thought to be required on ceramide molecules for the activation. Chymotrypsin-treated neutrophils, from which most L-selectin had been removed, reacted with sulfatide and galactocerebroside to enhance phagocytosis. These results suggest that an unidentified receptor for these cerebrosides exists on neutrophils and participates in the enhancement of phagocytosis.

Topics: Cell Adhesion; Cell Membrane; Cell Separation; Cells, Cultured; Ceramides; Cerebrosides; Chymotrypsin; Dose-Response Relationship, Drug; Flow Cytometry; Galactosylceramides; Glucosylceramides; Humans; Inflammation; L-Selectin; Macrophage-1 Antigen; Neutrophils; Phagocytes; Phagocytosis; Receptors, IgG; Sulfoglycosphingolipids

Extraction of control human spleen glucocerebrosidase with sodium cholate and butan-l-ol reversibly inactivates the enzyme in terms of its ability to hydrolyse the water-soluble substrate 4-methylumbelliferyl beta-D-glucopyranoside (MUGlc). The acidic brain lipid galactocerebroside 3-sulphate (sulphatide) reconstitutes beta-glucosidase activity in a strongly concentration-dependent manner. In this study we show that sulphatide exhibits three critical micellar concentrations (CMCs): CMC1, 3.72 microM; CMC2, 22.6 microM; CMC3, 60.7 microM. We designate the aggregates formed at these CMCs as primary, secondary and tertiary micelles respectively. From the results of kinetic studies performed at various sulphatide concentrations (0.012-248 microM), we found that sulphatide monomers (less than 3 microM) decreased the Km (for MUGlc) of control glucocerebrosidase from 11 to 4.6 mM, and lowered the Vmax. 2-fold. However, secondary and tertiary micelles were required for expression of high control glucocerebrosidase activities. Glucocerebrosidase prepared from the spleen of a patient with non-neuronopathic type 1 Gaucher's disease exhibited a very low Km (2.8 mM) even in the absence of exogenous lipid, and sulphatide monomers had no effect on the mutant enzyme's Km or Vmax. However, secondary or tertiary micelles markedly increased the Vmax. of the type 1 glucocerebrosidase to 60% of the corresponding control enzyme value. In contrast, for the glucocerebrosidase of the neuronopathic type 2 case, although sulphatide decreased the Km from 9.2 to 1.7 mM, the Vmax. never reached more than 5% that of the control enzyme, even at high concentrations of sulphatide. In addition, we found that secondary and tertiary sulphatide micelles enhanced the rate of inactivation of all three glucocerebrosidase preparations by chymotrypsin. Collectively, these results indicate the presence of two sulphatide-binding sites on glucocerebrosidase: one that enhances substrate binding, and another that enhances catalysis.

Topics: Binding Sites; Cerebrosides; Chymotrypsin; Enzyme Activation; Galactosylceramides; Gaucher Disease; Glucosidases; Glucosylceramidase; Glycoproteins; Humans; Kinetics; Micelles; Proteins; Saposins; Spleen; Sulfoglycosphingolipids