Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus.
Conglutinin is a bovine serum protein which was first described as a vertebrate lectin. This protein belongs to the family of C-type lectins. These lectins are composed of four characteristic domains: (1) an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and (4) a carbohydrate recognition domain (CRD). Recently lectins have been shown to function as immunoglobulin-independent defence molecules due to a complement-mediated mechanism or opsonization. Our previous study showed that bovine conglutinin can inhibit haemagglutination by influenza A viruses and act by directly neutralizing them due to its lectin properties. In order to elucidate the biological role of the collagen-like domain, a recombinant partial conglutinin lacking this collagen-like domain was produced in an Escherichia coli system and its biological activities were examined. A 497 bp sequence, consisting of a short collagen region (two repeats of G-X-Y amino acid sequences), the neck domain, and the CRD of conglutinin cDNA, was amplified by the reverse-transcriptase PCR technique. The cDNA was transferred to a bacterial expression vector system (pRSET-A) and stable transfectants with a high level of conglutinin production were obtained. SDS/PAGE and Western blotting analyses showed a recombinant fusion protein of 27 kDa. Results of a cross-linking study and gel-filtration assay indicated that the recombinant conglutinin can form a trimeric structure and that it has sugar binding activity and specificity similar to that of native conglutinin. The recombinant conglutinin was also found to inhibit haemagglutination caused by influenza A virus as well as to possess less conglutination activity. These results suggest that in order for conglutinin to inhibit haemagglutination caused by the influenza virus, as well as to have sugar binding activity or to form trimers, it does not require the N-terminal and collagenous domains; however, they are essential for full conglutination activity.
Eda
S
S
Osaka Prefectural Institute of Public Health, Department of Pathology, Japan.
Suzuki
Y
Y
Kase
T
T
Kawai
T
T
Ohtani
K
K
Sakamoto
T
T
Kurimura
T
T
Wakamiya
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003094:Collagen / Q000737:chemistry; D037222:Collectins; D003168:Complement Fixation Tests; D003432:Cross-Linking Reagents; D017931:DNA Primers; D004926:Escherichia coli; D006384:Hemagglutination / Q000187:drug effects*; D009980:Influenza A virus / Q000276:immunology*; D037102:Lectins; D008351:Mannans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012712:Serum Globulins / Q000737:chemistry* / Q000494:pharmacology / Q000502:physiology*; D012756:Sheep
D037222:Collectins; D003432:Cross-Linking Reagents; D017931:DNA Primers; D037102:Lectins; D008351:Mannans; D011994:Recombinant Proteins; D012712:Serum Globulins; C024014:conglutinin; D003094:Collagen