Mannan binding

Abstract: Recent identification of a C3-like gene in sea urchins revealed the presence of a complement system in invertebrates. To elucidate further the components and function of the pre-vertebrate complement system, we attempted to isolate an ascidian (urochordata) C3 convertase. After identification of C3 cDNA from Halocynthia roretzi, a Japanese ascidian, reverse transcriptase–PCR amplification of hepatopancreas RNA was performed using primers encoding highly conserved amino acid sequences of the vertebrate Bf and C2 serine protease domain. Two candidate sequences were identified, and the corresponding cDNA clones were isolated from a hepatopancreas library. Surprisingly, neither clone is related to Bf/C2 but rather share the same domain structure of mammalian C1r/C1s/MASP (mannan binding protein-associated serine protease), and are more related evolutionarily to mammalian MASP than to mammalian C1r or C1s. The identification of the tunicate MASP clones, amplified with primers designed to amplify Bf or C2, suggests that the lectin pathway antedated the classical and alternative pathways of complement activation.

Abstract: A rat liver mannan-binding protein was isolated by affinity chromatography on invertase--Sepharose by a modification of the method of Kawasaki, Etoh & Yamashina [(1978) Biochem. Biophys. Res. Commun. 81, 1018-1024] and by a new method involving chromatography on mannose-Sepharose. The binding protein appears as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent mol.wt. of approx. 30000. Binding of 125I-labelled mannan is saturable and inhibited by mannose, N-acetylglucosamine, or L-fucose but not by galactose or mannose 6-phosphate. Neoglycoproteins containing mannose, N-acetylglucosamine, or L-fucose, but not galactose, are inhibitory. The neoglycoproteins are 10000-fold more effective (based on moles of sugar) than are free monosaccharides as inhibitors. 125I-labelled mannan binding to the binding protein is calcium-dependent.

Abstract: Low plasma concentration of mannan binding protein (MBP) has been shown to be the basis for a common opsonic deficiency and suggested to be caused by a single nucleotide substitution at base 230 of exon 1 in the MBP gene. This substitution causes a replacement of glycine (codon GGC) with aspartic acid (codon GAC). Of 123 healthy Danish individuals investigated by polymerase chain reaction performed on exon 1, followed by restriction fragment length polymorphism or allospecific probing, 93 were homozygous (75.6%) for GGC, 28 heterozygous (22.8%), and two homozygous for GAC (1.6%). The gene frequency of the GAC allele was found to be 0.13. DNA sequencing of the cloned exon 1 from one GAC homozygous individual revealed no other substitution. The median MBP concentration in the group containing the GAC allele was 6.4 times lower than in the GGC homozygous group (195 and 1234 micrograms/l respectively). However, the range in plasma concentrations of MBP was wide and overlapping between the groups. MBP protein was detected in both the GAC homozygotes (9 and 387 micrograms/l). Furthermore, no difference in relative mass and biological activity (mannan binding) was found when sera containing the two forms of MBP were investigated. Accordingly, it can be concluded that the GAC allele is able to produce a functional MBP protein which may be detected in serum at low concentrations.

Abstract: Rat monoclonal antibodies (MoAbs) against mouse mannan-binding lectin (MBL)-A and MBL-C were generated and assays for MBL-A and MBL-C were constructed. This allowed for the quantitative analysis of both proteins for the first time. Previously only MBL-A has been quantified using less standardized methods. In a mouse serum pool the concentrations were now determined at 7.5 µg MBL-A and 45 µg MBL-C per ml. On gel permeation chromatography of mouse serum, MBL-A eluted corresponding to a Mr of 850 kDa whereas the majority of MBL-C eluted corresponding to a Mr of 950 kDa. On sucrose density gradient centrifugation the sedimentation velocities of MBL-A and MBL-C were estimated at 7.3 S and 10.8 S, respectively. The MBL-A and MBL-C levels in 10 laboratory mice strains were compared and found to vary between 4 µg/ml to 12 µg/ml, and 16 µg/ml to 118 µg/ml, respectively. After the induction of acute phase responses by intraperitoneal injection of either casein or lipopolysaccharide (LPS), MBL-A was found to increase approximately two-fold, with a maximum after 32 h, while MBL-C did not increase significantly. In comparison, serum amyloid A component (SAA) peaked at 15 h with an approximate 100-fold increase.