Myosin II molecules assemble and form filaments through their C-terminal rod region, and the dynamic filament assembly-disassembly process of nonmuscle myosin II molecules is important for cellular activities. To estimate the critical region for filament formation of vertebrate nonmuscle myosin II, we assessed the solubility of a series of truncated recombinant rod fragments of nonmuscle myosin IIB at various concentrations of NaCl. A C-terminal 248-residue rod fragment (Asp 1729-Glu 1976) was shown by its solubility behavior to retain native assembly features, and two regions within it were found to be necessary for assembly: 35 amino acid residues from Asp 1729 to Thr 1763 and 39 amino acid residues from Ala 1875 to Ala 1913, the latter containing a sequence similar to the assembly competence domain (ACD) of skeletal muscle myosin. Fragments lacking either of the two regions were soluble at any NaCl concentration. We referred to these two regions as nonmuscle myosin ACD1 (nACD1) and nACD2, respectively. In addition, we constructed an alpha-helical coiled-coil model of the rod fragment, and found that a remarkable negative charge cluster (termed N1) and a positive charge cluster (termed P2) were present within nACD1 and nACD2, respectively, besides another positive charge cluster (termed P1) in the amino-terminal vicinity of nACD2. From these results, we propose two major electrostatic interactions that are essential for filament formation of nonmuscle myosin II: the antiparallel interaction between P2 and N1 which is essential for the nucleation step and the parallel interaction between P1 and N1 which is important for the elongation step.

No abstract

No abstract

Three novel missense mutations in the human lysosomal sialidase gene causing amino acid substitutions (P80L, W240R, and P316S) in the coding region were identified in two Japanese sialidosis patients. One patient with a severe, congenital form of type 2 sialidosis was a compound heterozygote for 239C-to-T (P80L) and 718T-to-C (W240R). The other patient with a mild juvenile-onset phenotype (type 1) was a homozygote for the base substitution of 946C-to-T (P316S). None of these mutant cDNA products showed enzymatic activity toward an artificial substrate when coexpressed in galactosialidosis fibroblastic cells together with protective protein/cathepsin A (PPCA). All mutants showed a reticular immunofluorescence distribution when coexpressed with the PPCA gene in COS-1 cells, suggesting that the gene products were retained in the endoplasmic reticulum/Golgi area or rapidly degraded in the lysosomes. Homology modeling of the structural changes introduced by the mutations predicted that the P80L and P316S transversions cause large conformational changes including the active site residues responsible for binding the sialic acid carboxylate group. The W240R substitution was deduced to influence the molecular surface structure of a limited region of the constructed models, which was also influenced by previously identified V217M and G243R transversions.

No abstract