Functional Characterization of Three Sialidase Proteins From Schaalia odontolytica

Abstract

<p><strong>Objectives: </strong></p><p>Sialidases (neuraminidases) are glycosidases responsible for removing terminal sialic acid units from glycoconjugates. They are considered potential virulence factors that promote oral bacteria colonization. Schaalia odontolytica (formerly Actinomyces odontolyticus), a primary colonizer of the oral cavity, encodes three sialidase homologues, Sia1 (338 aa, 35 kDa), Sia2 (740 aa, 79 kDa) and Sia3 (745 aa, 81 kDa). This study aimed to characterize and compare the biochemical functions of Sia1-3 from S. odontolytica ATCC 17929^T.<br></p><p><strong>Methods:</strong></p><p>Full-length Sia1-3, and truncations of Sia2 (Sia2-cat; residues 171-552) and Sia3 (Sia3-cat; residues 275-745) that respectively comprised the predicted catalytic domains, were cloned, expressed and purified as N-terminal His-tagged proteins using standard techniques. The hydrolytic activities of the recombinant sialidases were assessed using the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (MUNANA). The substrate specificity of the sialidases was quantified using sodium periodate/thiobarbituric acid (TBA) assays with sialyl- α2,3-lactose and sialyl- α2,6-lactose substrates.<br></p><p><strong>Results:</strong></p><p>All three sialidases showed optimal activities under acidic conditions (ca. pH 4.5-6.5). Kinetic analysis of MUNANA hydrolysis indicated Sia2 had the lowest KM value (0.06 ± 0.01 mM), highest kcat value (6.8 ± 0.5 s-1), making its catalytic efficiency ca. 15 to 20-fold higher than that of Sia1 and Sia3. The catalytic efficiency of Sia2-cat was ca. 13-fold higher than that of Sia2. In contrast, Sia3-cat exhibited negligible hydrolytic activities. Sia2 was more sensitive than Sia1 and Sia3 to two sialidase inhibitors: 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en) and siastatin B. Sia1-3 were all capable of cleaving α-2,3- and α-2,6-linkages, albeit with varying selectivities.<br></p><p><strong>Conclusions:</strong></p><p>These results indicate that the respective Sia1-3 proteins have notably different biochemical properties and activities, suggesting they play distinct biological roles in S. odontolytica. Notably, the non-catalytic domains of Sia2 play modulatory roles that regulating its sialidase activities. Investigations into the biological functions of Sia1-3 are ongoing.<br></p>