Structural characterization of bovine collectin-43.
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 +/- 0.1 kDa. Furthermore, the mass spectrum showed the presence of a truncated version of the polypeptide, which has also previously been shown by SDS/PAGE 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, 11820-11824] 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 O-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 Cys15 in polypeptide chain 1 to Cys15 in polypeptide chain 2, Cys20 in chain 2 to Cys20 in chain 3 and Cys20 in chain 1 to Cys15 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.
Rothmann
A B
AB
Department of Molecular Biology, University of Odense, Denmark.
Mortensen
H D
HD
Holmskov
U
U
Højrup
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D037222:Collectins; D004220:Disulfides / Q000737:chemistry; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012712:Serum Globulins / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D037222:Collectins; D004220:Disulfides; D037102:Lectins; D010455:Peptides; D012712:Serum Globulins; C086954:collectin-43 protein, bovine