Revealing the Effects of Missense Mutations Causing Snyder-Robinson Syndrome on the Stability and Dimerization of Spermine Synthase

Abstract

Missense mutations in spermine synthase (SpmSyn) protein have been shown to cause the Snyder-Robinson syndrome (SRS). Depending on the location within the structure of SpmSyn and type of amino acid substitution, different mechanisms resulting in SRS were proposed. Here we focus on naturally occurring amino acid substitutions causing SRS, which are situated away from the active center of SpmSyn and thus are not directly involved in the catalysis. Two of the mutations, M35R and P112L, are reported for the first time in this study. It is demonstrated, both experimentally and computationally, that for such mutations the major effect resulting in dysfunctional SpmSyn is the destabilization of the protein. In vitro experiments indicated either no presence or very little amount of the mutant SpmSyn in patient cells. In silico modeling predicted that all studied mutations in this work destabilize SpmSyn and some of them abolish homo-dimer formation. Since dimerization and structural stability are equally important for the wild type function of SpmSyn, it is proposed that the SRS caused by mutations occurring in the N-domain of SpmSyn is a result of dysfunctional mutant proteins being partially unfolded and degraded by the proteomic machinery of the cell or being unable to form a homo-dimer.

Keywords: Snyder-Robinson syndrome; binding affinity charge; energy calculations; missense mutation; spermine synthase.

Figure 1

Western blot analysis of SMS levels in patient lymphoblast cell lines. (A) Denatured SMS blot. 10 µg of lymphoblast lysate was prepared in Lamelli sample buffer. The buffer was parted on a 4%–20% sodium dodecyl sulfate polyacrylamide gel (SDS–PAGE). Furthermore the buffer was probed for SMS and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The control was GAPDH. Densitometry of the blots was analyzed using NIH Image J. SMS expression levels of the mutants were normalized to the control; (B) Native SMS blot. 10 µg of lymphoblast cell lysate was prepared in native sample buffer, separated on a native PAGE gel and probed for SMS and GAPDH. Densitometry of the blots was analyzed by NIH Image J.

Figure 2

Sequence alignment of SpmSyn among different species. The mutation sites considered in this study are represented in bold letters and the position of the residue in human SpmSyn is shown at the top. The mammals considered in this study are represented in bold letters. Multiple sequence alignment (MSA) is performed with Cobalt Constraint-based Multiple Protein Alignment Tool (COBALT).

Figure 3

The side chain conformation of five disease-causing mutations mapped onto SpmSyn: (a) Part of SpmSyn zoomed at WT position of Met35; (b) Part of SpmSyn zoomed at MT position of Arg35; (c) Part of SpmSyn zoomed at WT position of Gly56; (d) Part of SpmSyn zoomed at MT position of Ser56; (e) Part of SpmSyn zoomed at WT position of Phe58; (f) Part of SpmSyn zoomed at MT position of Leu58; (g) Part of SpmSyn zoomed at WT position of Gly67; (h) Part of SpmSyn zoomed at MT position of Glu67; (i) Part of SpmSyn zoomed at WT position of Pro112; (j) Part of SpmSyn zoomed at MT position of Leu112; The side chain of WT and MT position is shown in red. Two different chains of the dimer are shown in blue and green.