Purification, characterization, cDNA cloning, and expression of asialofetuin-binding C-type lectin from eggs of shishamo smelt (Osmerus [Spirinchus] lanceolatus).
A novel C-type lectin (OLABL) was isolated from the eggs of shishamo smelt [Osmerus (Spirinchus) lanceolatus] by affinity chromatography on asialofetuin-Sepharose. OLABL had a molecular mass of 29 kDa on SDS-PAGE under nonreducing conditions and two subunits with masses of 15 kDa (OLABL-H) and 14 kDa (OLABL-L) under reducing conditions. Thus, OLABL is a heterodimeric protein. cDNA sequence analysis revealed that the H- and L-subunits of OLABL were composed of 137 and 136 amino acid residues, respectively, and showed almost identical (95%) sequences, with slight differences in the N-terminal and C-terminal regions. Since each subunit contained only the characteristic motif of C-type lectin-like domain (CTLD), EPN-E-WND, OLABL is a member of group VII of the CTLD-containing protein family. Although OLABL had an EPN sequence that is known as a mannose-specific motif found in the collectin family, OLABL agglutinated rabbit erythrocytes without the addition of Ca(2+) ion, and this activity was inhibited by l-rhamnose and d-galactose derivatives, but not by d-mannose and d-glucose. These results indicate that OLABL has similar characteristics to AJL-2, a calcium-independent lactose specific lectin isolated from Japanese eel skin mucus. Recombinant OLABLs (rHisOLABLs), His-tagged homodimers of the H- and L-subunits, were refolded from inclusion bodies expressed by Escherichia coli. rHisOLABL-L was recovered as a soluble form, but rHisOLABL-H was hardly dissolved in a renaturing buffer. The specific activities of rHisOLABL-L, rHisOLABL-H, and native OLABL were 500, 36, and 20, respectively. These findings suggest that the combination of subunits may affect the solubility and activity of these dimeric form lectins.
Hosono
Masahiro
M
Cancer Research Institute, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan.
Sugawara
Shigeki
S
Ogawa
Yukiko
Y
Kohno
Takayuki
T
Takayanagi
Motoaki
M
Nitta
Kazuo
K
D000371:Agglutination / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004527:Egg Proteins / Q000737:chemistry / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects* / Q000276:immunology*; D060748:Fetuins; D006801:Humans; D037181:Lectins, C-Type / Q000008:administration & dosage* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D036441:Osmeriformes / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D000509:alpha-Fetoproteins / Q000378:metabolism*
D001212:Asialoglycoproteins; D018076:DNA, Complementary; D004527:Egg Proteins; D060748:Fetuins; D037181:Lectins, C-Type; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D002118:Calcium