Abstract
Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile(170), Ser(172), and Ser(226) with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys(30), located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi.
Highlights
Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting
In this study we examined the characteristics of dimer formation observed for the soluble and membrane-bound forms of the human calcium activated nucleotidases (CAN) protein
In the work examining the soluble, secreted SCAN expressed by mammalian COS cells, the size exclusion chromatography used to examine the apparent size of the protein was performed in a buffer containing 5 mM Ca2ϩ and 100 mM NaCl (2)
Summary
These vertebrate nucleotidases may play a role in protein glycosylation The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. Because of its substrate preference and subcellular localization, Failer et al (1) postulated that the membrane-bound rat enzyme may function to support glycosylation reactions related to protein biosynthetic quality control in the endoplasmic reticulum These mammalian enzymes are termed calcium activated nucleotidases (CAN),[3] as described in a recent review (8). The reason for the observed increased nucleotidase activities of a previously published mutation of SCAN (E130Y) (9) was not clear, given that this residue is far removed from the active site as identified by the crystal structure of the human enzyme co-crystallized with a nonhydrolyzable nucleotide analogue (7). Our results indicate that the interface of the dimer is localized primarily within the 2d and 3d elements of the -propeller and that the dimer interface is dominated by hydrophobic interactions
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