Efficient silica synthesis from tetra(glycerol)orthosilicate with cathepsin- and silicatein-like proteins

www.nature.com/scientificreports OPEN Received: 28 March 2018 Accepted: 24 October 2018 Published: xx xx xxxx Efficient silica synthesis from tetra(glycerol)orthosilicate with cathepsin- and silicatein-like proteins Natalia V. Povarova1, Nikolay A. Barinov2, Mikhail S. Baranov1,3, Nadezhda M. Markina1, Anna M. Varizhuk2, Galina E. Pozmogova2, Dmitry V. Klinov2, Valery B. Kozhemyako4 & Konstantin A. Lukyanov1 Silicateins play a key role in biosynthesis of spicules in marine sponges; they are also capable to catalyze formation of amorphous silica in vitro. Silicateins are highly homologous to cathepsins L – a family of cysteine proteases. Molecular mechanisms of silicatein activity remain controversial. Here site-directed mutagenesis was used to clarify significance of selected residues in silica polymerization. A number of mutations were introduced into two sponge proteins – silicatein A1 and cathepsin L from Latrunculia oparinae, as well as into human cathepsin L. First direction was alanine scanning of the proposed catalytic residues. Also, reciprocal mutations were introduced at selected positions that differ between cathepsins L and silicateins. Surprisingly, all the wild type and mutant proteins were capable to catalyze amorphous silica formation with a water-soluble silica precursor tetra(glycerol)orthosilicate. Some mutants possessed several-fold enhanced silica-forming activity and can potentially be useful for nanomaterial synthesis applications. Our findings contradict to the previously suggested mechanisms of silicatein action via a catalytic triad analogous to that in cathepsins L. Instead, a surface-templated biosilification by silicateins and related proteins can be proposed. Silicateins are the common spicule-forming proteins of marine sponges1. They are homologous to cathepsins – a family of proteases acting mainly in lysosomes at the low pH2. Most cathepsins are cysteine proteases with Cys-His-Asn catalytic triad, although some cathepsins are serine or aspartate proteases. Among different cathepsin groups, cathepsins L are the closest homologs of silicateins. In spite of the evolutionary and functional divergence, sponge silicateins share over 50% identity with human cathepsin L (CTSL). Amino acid alignment shows that the active site amino acids His and Asn are conserved, whereas position 26 (25 for CTSL) is occupied by Cys in CTSL and Ser in silicateins1. This Cys to Ser replacement is thought to be the key functional difference between the two protein classes. During spicule formation in sponges, silicateins interact with few more proteins, such as collagen, galectin, and silintaphins, and form long fibers, which promote silica polymerization around them3. Silicatein-associated proteins have an impact on the silicatein activity4, but silicatein itself is sufficient to form amorphous5,6 silica from different silica acid precursors in vitro. Catalytic mechanism of the silicateins is not directly confirmed, but the key step is supposed to be a binding and activation of silica acid or silica acid precursor by the enzyme active site. Similarly to highly homologous CTSL, the active site is formed by Ser25 and His163. It is generally thought that these residues activate silicon atom of the substrate which results in hydrolysis (Fig. 1A)7 or polymerization (Fig. 1B)8, or both9. The exact molecular mechanism of interactions between the active site residues and substrate remains controversial. Some mechanisms suggest formation of the covalent bond between silicon atom and Ser26 (Fig. 1A,B)9, other include extra stabilization by Gln residue without covalent binding (Fig. 1C)8. Cysteine in the active site is supposed 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997, Moscow, Russia. Federal Research and Clinical Center of Physical-Chemical Medicine, Malaya Pirogovskaya 1a, 119435, Moscow, Russia. 3Pirogov Russian National Research Medical University, Ostrovitianov 1, 117997, Moscow, Russia. 4G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences, 690022, Vladivostok, Russia. Correspondence and requests for materials should be addressed to K.A.L. (email: ) 2 Scientific Reports | (2018) 8:16759 | DOI:10.1038/s41598-018-34965-9 1 www.nature.com/scientificreports/ Figure 1. Previously proposed mechanisms of the silicatein activity. (A) Tetraethyl orthosilicate (TEOS) hydrolysis by silicatein7. (B,C) Two proposed mechanisms of silica acid condensation8,9. to be non-efficient for silica polymerization catalysis and is commonly described as a feature of cathepsins L3. Site-directed mutagenesis confirmed the crucial role of Ser26 and His165 in silicateins7,10. However, two recent works demonstrated that in spite of the presence of Cys in the active site, cathepsin L can possess silica-polymerizing activity in some conditions11,12. We hypothesized that previously described effects of substitutions of the catalytic residues were overestimated due to the suboptimal silica acid precursor used. Recently, a highly water soluble silica acid precursor tetra(glycerol)orthosilicate (TGS) was introduced that provides a convenient and efficient way to assess protein activity13. Here, to clarify the role of certain amino acid residues, activity of silicatein and cathepsin L mutants with TGS was systematically evaluated. Results Protein isolation and purification. Usually bacterially expressed silicateins are poorly soluble and require some extra tags to be produced in a soluble form7. Genes of two wild-type proteins from marine sponge Latrunculia oparinae were cloned, namely silicatein A1 (LoSilA1) and cathepsin L (LoCath), in several vectors, and different expression systems to express proteins were tested: pQE30 in XLBlue and BL21 strains, pBAD in BW and XJB strains, pET-40b(+) in XJB, BL and BL21codon+ strains. Also, different temperature (room temperature and 37 °C) and different concentration of inductors were tested. The conditions described in experimental section was the only one, which allowed to express proteins in soluble fraction. LoSilA1 and LoCath were still difficult to purify from the cell lysate by metal-affinity chromatography until His-tag was relocated to the C-terminus. The yield of the protein was about 1 mg per 1 liter of the cell culture. The purity of the proteins (more than 85%) was confirmed by the SDS-PAGE (representative example is shown in Fig. 2). Due to high protein similarity the same conditions were used for CTSL, but it was found that this protein should be expressed on 25 °C after induction. To verify protein functionality scanning electron microscopy (SEM) was used. The data showed that both LoSilA1 and LoCath form similar amorphous silica particles with TGS (Fig. 3). Mutagenesis of sponge Latrunculia oparinae proteins. Point mutations of the catalytic residues and their close neighbourhoods were introduced. Residues at positions 20, 26, (...truncated)