Purification, characterization, cDNA cloning, and expression of asialofetuin-binding C-type lectin from eggs of shishamo smelt ( Osmerus [ Spirinchus] lanceolatus)

• Published in: Biochimica et biophysica acta Vol. 1725; no. 2; pp. 160 - 173
• Main Authors: Hosono, Masahiro, Sugawara, Shigeki, Ogawa, Yukiko, Kohno, Takayuki, Takayanagi, Motoaki, Nitta, Kazuo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.09.2005
• Subjects: Agglutination - drug effects; alpha-Fetoproteins - metabolism; Amino Acid Sequence; Animals; Asialofetuin; Asialoglycoproteins - metabolism; Binding Sites; C-type lectin; Calcium - metabolism; Calcium-independent; Cells, Cultured; Cloning, Molecular; DNA, Complementary - genetics; Dose-Response Relationship, Drug; Egg Proteins - chemistry; Egg Proteins - pharmacology; EPN sequence; Erythrocytes - drug effects; Erythrocytes - immunology; Fetuins; Fish egg; Heterodimer; Humans; Lectins, C-Type - administration & dosage; Lectins, C-Type - metabolism; Molecular Sequence Data; Molecular Weight; Osmeriformes - metabolism; Protein Binding; Rabbits; Recombinant Proteins - chemistry; Recombinant Proteins - pharmacology; Sequence Homology, Amino Acid; Ctr; 2-ME; EPN sequence; HU; Ntr; Heterodimer; BSA; Calcium-independent; Asialofetuin; RTase; Fish egg; RBL; C-type lectin; aa; PBS; OLRBL; TBS; GSP; SDS-PAGE; OLABL; PB; CRD; PMSF; SEF; LB; RACE; IPTG; AUAP
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2005.07.009

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.