Yasunori Naganawa

Molecular and structural studies of Japanese patients with sialidosis type 1

AbstractTo gain insight into the pathogenesis of sialidosis type 1, we performed molecular investigations of two unrelated Japanese patients. Both of them are compound heterozygotes for base substitutions of 649G-to-A and 727G-to-A, which result in amino acid alterations V217M and G243R, respectively. Using homology modeling, the structure of human lysosomal neuraminidase was constructed and the structural changes caused by these missense mutations were deduced. The predicted change due to V217M was smaller than that caused by G243R, the latter resulting in a drastic, widespread alteration. The overexpressed gene products containing these mutations had the same molecular weight as that of the wild type, although the amounts of the products were moderately decreased. A biochemical study demonstrated that the expressed neuraminidase containing a V217M mutation was partly transported to lysosomes and showed residual enzyme activity, although a G243R mutant was retained in the endoplasmic reticulum/Golgi area and had completely lost the enzyme activity. Considering the data, we surmise that the V217M substitution may be closely associated with the phenotype of sialidosis type 1 with a late onset and moderate clinical course.

Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes

AbstractThree 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.

Generation of mouse-human hybridomas secreting antibodies against peanut allergen Ara h1.

Two clones of mouse–human hybridomas, secreting human monoclonal antibodies to a peanut allergen Ara h1, were generated from human peripheral blood lymphocytes transformed with Epstein--Barr virus, followed by cell fusion with mouse myeloma cells. Epitope analysis with overlapping peptides synthesized on a multi-pin apparatus revealed antibody-binding sequences of Ara h1 protein.

Epitope analysis of Japanese cedar pollen allergen Cry j2 with a human IgM class monoclonal antibody 404-117

Japanese cedar pollen allergen Cry j2 is a causal allergen of seasonal pollinosis in Japan. To analyze B cell epitopes of Cry j2, we established two human-mouse hybridomas secreting IgM class human monoclonal antibodies to Cry j2. A pin-peptide enzyme-linked immunosorbent assay with synthesized icosa peptides showed that 404-117 monoclonal antibody bound to peptides #11-13 with cry j2 amino acid sequence of 101F-L140. Detailed analysis with octa peptides and alanine substituted peptides indicated that an amino acid sequence of 118FKVD121 was an essential for antibody binding. When K119 (Asn) was substituted with alanine, 404-117 monoclonal antibody did not bind to the alanine substituted peptide. We concluded that the 118FKVD121 sequence might have a very important role in early recognition by Cry j2-specific B cells, which could act as antigen presenting cells.