The mutant hen egg white lysozymes Ile55Thr and Asp66His, corresponding to human amyloidogenic mutant lysozymes Ile56Thr and Asp67His, respectively, were secreted in Saccharomyces cerevisiae. The amyloidogenic mutants (I55T and D66H) of hen egg white lysozymes were remarkably less soluble than that of the wild-type protein. To enhance the secretion of these mutants, we constructed the glycosylated amyloidogenic lysozymes (I55T/G49N and D66H/G49N) having the N-glycosylation signal sequence (Asn-X-Ser) by the substitution of glycine with asparagine at position 49. The secretion of these glycosylated mutant proteins is greatly increased in S. cerevisiae, compared with that of non-glycosylated type. Both the glycosylated mutants retained about 40% enzymatic activity when incubated at pH 7.4 for 1 h at the physiological temperature of 37³C whereas the non-glycosylated proteins eventually lost all activity under these conditions. These results suggest that the glycosylated chains could mask the L L-strand of amyloidogenic lysozymes from the intermolecular cross-L L-sheet association, thus improving the solubility of amyloidogenic lysozymes. ß

No abstract

Pokeweed lectin (PL), a lectin specific for N-acetylglucosamine-containing saccharides, stimulates peripheral lymphocytes to undergo mitosis by binding to their cell surfaces. Four types of lectins have been isolated from the roots of pokeweed (Phytolacca americana) and shown to contain homologous domains but to have different molecular sizes and biological properties. PL-D, the smallest lectin in the group, has two isolectins, PL-D1 and PL-D2. PL-D1 consists of 84 amino-acid residues, while PL-D2 is identical to PL-D1 in sequence except for the lack of two C-terminal residues, Leu83 and Thr84. The crystal structures of PL-D1 and PL-D2 were solved by the molecular-replacement method and refined to 1.65 and 1.5 A resolution with R factors of 17.2 and 17.6%, respectively. The PL-Ds are composed of two repetitive chitin-binding domains, each of which has four S-S bridges and one putative carbohydrate-binding site. The two carbohydrate-binding sites in PL-D are located on one side of the molecule. The relative orientation of the two domains in PL-D1 differs from that in PL-D2. Two C-terminal residues of PL-D1 are invisible in the present crystal structure, indicating the flexibility of the region. PL-D2 has a Ca2+ ion bound to the C-terminus on the molecular surface. A wide distribution of acidic residues is characteristically observed on one side of the C-terminal region of PL-D.

Lectin C from the roots of pokeweed (Phytolacca americana) (PL-C; 13 747 Da, 126 amino-acid residues), which consists of three chitin-binding domains, was initially crystallized in two crystal forms. One form, obtained in the presence of 30%(w/v) PEG 4000, belongs to the tetragonal system. The other, obtained in the presence of 2.0 M ammonium sulfate, belongs to the rhombohedral system. Statistical analyses of the X-ray diffraction intensities showed that both crystals were twinned. Single crystals suitable for a diffraction experiment were obtained by the addition of 0.5%(v/v) dioxane to the latter precipitant solution. They belong to space group R3, with unit-cell parameters a = b = 104.1, c = 69.7 A, and diffract X-rays to 1.8 A resolution. A heavy-atom derivative crystal has been obtained and structure determination is presently ongoing using the SIRAS method.

No abstract