Characterization of calcium binding properties of lithostathine

Abstract.

The pancreas secretes primarily two types of metabolically important proteins: digestive enzymes and hormones. Lithostathine (LIT) is the only protein excreted from the pancreas that has no known digestive or hormonal activity. Human lithostathine is a 144-amino acid glycoprotein synthesized by the exocrine pancreas that has been implicated in various physiological functions, including inhibition of pancreatic stone formation. To better understand the physiological function of LIT, we expressed the recombinant LIT protein in Escherichia coli and measured its calcium binding properties by equilibrium dialysis and electron paramagnetic resonance (EPR) spectroscopy. Equilibrium dialysis with ⁴⁵Ca²⁺ showed that LIT binds Ca²⁺ with 1:1 stoichiometry. EPR studies using the divalent vanadyl (VO²⁺) ion as a paramagnetic substitute for Ca²⁺ also showed that VO²⁺ binds to LIT with a metal:protein binding stoichiometry of 1:1 and that VO²⁺ competes with Ca²⁺ in binding to LIT. Mutations of a cluster of acidic residues on the molecular surface (E30A, D31A, E33A, D37A, D72A, and D73A) resulted in almost complete loss (95–100%) of binding of Ca²⁺ and VO²⁺, showing that these residues are critical for calcium binding by LIT.