Bovine collectin-43 (CL-43), the most recently disclosed member of the collectin group, has been characterized structurally at the protein level by a combination of mass spectrometry and protein sequencing. The molecular mass of reduced CL-43 was determined by the use of mass spectrometry to be 33.6 2 0.1 kDa. Furthermore, the mass spectrum showed the presence of a truncated version of the polypeptide, which has also previously been shown by SDSPAGE and N-terminal sequencing. N-terminal Edman degradation of peptides from a tryptic digestion of native CL-43 verified the published sequence derived from cDNA studies and partial protein sequencing [Lim, B.-L., Willis, A. C., Reid, K. B. M., Lu, J., Lauersen, S. B., Jensenius, J. C. & Holmskov, U. (1994) J. Biol. Chem. 269, 11 820-11 8241 with two exceptions. Using mass spectrometry and N-terminal sequencing, a large number of post-translational modifications were found in the collagen-like region (repetitive Gly-Xaa-Yaa sequence). All proline residues located in the Yaa-position in the collagen-like region were found to be partially hydroxylated while all lysine residues in the Yaa position were fully hydroxylated and glycosylated. The glycosylation was determined as glycosyl-galactosyl 0-linked to a hydroxylated lysine residue. Mass spectrometric analysis of a peptic digest of the N-terminal tryptic peptide revealed that the three polypeptide chains were disulphide linked in a rather surprising pattern. The cysteine residues were inter-chain disulphide linked by Cysl5 in polypeptide chain 1 to Cysl5 in polypeptide chain 2, Cys20 in chain 2 to Cys20 in chain 3 and Cys20 in chain 1 to Cysl5 in chain 3.The four cysteine residues at the C-terminus were intra-chain disulphide linked, Cys204 to Cys299 and Cys277 to Cys291, as expected for a C-type lectin.Keywords: C-type lectin; mass spectrometry; post-translational modifications; disulfide bonds; collagenlike.Bovine CL-43 belongs to a group of proteins called collectins, containing a collagenous region linked to a C-terminal carbohydrate-recognition domain (CRD) common to the family of calcium-dependent carbohydrate-binding proteins known as C-type animal lectins (Drickamer, 1988). Other members of this family are bovine conglutinin and mannan-binding lectin (MBL), which are serum proteins like CL-43, and surfactant protein A and surfactant protein D (SP-A and SP-D), purified from lung pulmonary surfactant of various species. All collectins are involved in the innate immune defence. MBL, conglutinin, SP-D and SP-A has been shown to bind directly to polysaccharides on various pathogens, and it has been proposed that their collagenous regions are ligands for the collectin receptor on phagocytes, thus mediating phagocytosis of coated micro-organism (Malhotra et al., 1990). In addition, bovine conglutinin causes opsonization by binding to glycoproteins derived from complement C3, notably iC3b (Hirani et al., 1985;Lauersen et al., 1994;Friis-Christiansen et al., 1990). MBL also has the ability to activate the classical compl...

The authors report on the purification and characterization of mannan- binding proteins (MBP) isolated from porcine serum. The MBPs were purified by use of PEG precipitation, affinity chromatography on mannan-Sepharose, protein A- and anti-porcine IgM-Sepharose followed by gel filtration. The MBP proteins were collagenase sensitive and showed gamma 1-gamma 2-electrophoretic mobility. The MBP designated pMBP-28 had a molecular mass of 28 kDa when analysed on SDS-PAGE under reducing conditions and eluted corresponding to a molecular mass of approximately 700 kDa on gel filtration chromatography. Electron micrographs of pMBP-28 revealed an oligomeric protein similar to rodent MBP-A and human MBP but with a predominance of penta- and hexameric molecules. Another protein designated pMBP-27 was composed of peptides of 27 kDa and had an Mr of 300-350 kDa on gel filtration chromatography. Electron microscopy of pMBP-27 showed dimer and trimer molecules; the trimers without distinct stalk regions. The N-terminal 26(pMBP-27) and 24(MBP-28) amino acid residues showed 54% and 58% identity with human MBP.pMBP-28 showed a higher degree of sequence similarity to rat and mouse MBP-A (60% identity) than to mouse and rat MBP-C (41-45% identity). Both pMBPs exhibited Ca2+-dependent binding to D-mannose immobilized on agarose but no significant binding to N-acetyl-D-glucosamine- or fucose-agarose. The results further suggested the presence of a third pMBP which copurified with pMBP-27 but this protein was not sequenced.

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