Laboratory of Immunobiology of Infections, Institute for Medical Biology, Polish Academy of Sciences, Lodowa 106, PL-93-232 Lodz, Poland
The ficolin-1 (M), -2 (L), -3 (H) and mannan-binding lectin (MBL) activate the complement system and have opsonic activity. The specificity of ficolin-3 is poorly characterised and currently limited to a few ligands only. We present new specific targets for human ficolin-3, identified among lipopolysaccharides (LPS, endotoxin) of Hafnia alvei. The interaction was restricted to LPSs of four strains: 23, PCM 1200, PCM 1203, PCM 1205 and limited to their O-specific polysaccharides (O-specific PS) composed of different number of oligosaccharide repeating units (RU). Moreover, these LPS/Ficolin-3 complexes activated the lectin pathway of complement in a C4b-deposition assay in calcium- and magnesium-dependent way.A neoglycoconjugate of the O-specific PS fraction of H. alvei 1200 LPS with BSA was prepared and used as a tool for determination of ficolin-3 concentration and activity in serum. To confirm a structure of the O-specific PS 1200 selected for the conjugate preparation, structural analysis was performed on a series of O-specific PSs released by mild acid hydrolysis of the LPS. The isolated O-specific PSs, showed the different length distribution, were devoid of a major part of core oligosaccharide region and had Hep-Kdo disaccharide at a reducing end. The neoglycoconjugate was a highly selective tool for determination of ficolin-3 concentration and activity in serum (lectin pathway activation in C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or natural antibodies.
13C NMR, 1H NMR, NMR-2D, SDS-PAGE, sugar analysis, mild acid hydrolysis, Western blotting, MALDI-TOF MS, serological methods, conjugation
interaction of ficolin-3 with H. alvei LPSs
Structure of the PS5 isolated by the mild acid hydrolysis of H. alvei 1200 LPS.
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7 C8
7,3,2,2 Ac
7,3,2,3 65%Ac
7,3,2 bDGlcpN 99.7 53.9 76.7 67.7 76.1 60.9
7,3,3,0,1,3,3,2,2 Ac
7,3,3,0,1,3,3,2,3 65%Ac
7,3,3,0,1,3,3,2 bDGlcpN 99.7 54.0 77.2 68.0 76.1 60.9
7,3,3,0,1,3,3,3,0,1,4 Ac
7,3,3,0,1,3,3,3,0,1 bDQuip4N 102.8 73.8 73.5 56.8 71.2 16.9
7,3,3,0,1,3,3,3,0 xDGro 70.8 69.5 66.5
7,3,3,0,1,3,3,3 P
7,3,3,0,1,3,3,4 aDGlcp 100.3 72.1 72.8 69.5 72.2 60.1
7,3,3,0,1,3,3 bDGalp 100.2 74.6 77.5 76.9 75.0 60.2
7,3,3,0,1,3,2 Ac
7,3,3,0,1,3 aDGlcpN 97.3 52.3 79.4 67.7 71.2 60.1
7,3,3,0,1,4 Ac
7,3,3,0,1 bDQuip4N 102.8 72.5 77.3 56.7 71.2 16.5
7,3,3,0 xDGro 70.8 69.5 66.5
7,3,3 P
7,3 bDGalp 100.8 75.1 78.6 67.7 74.9 60.8
7,2 Ac
7 bDGlcpN 100.4 56.8 82.5 68.7 ? 60.8
8 aXLDmanHepp 99.3 69.9 70.8 66.2 71.4 69.3 63.7
?XKdop 174 96.7 34.2 65.9 66.2 69.0 75.7 64.4
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7 H8
7,3,2,2 Ac
7,3,2,3 65%Ac
7,3,2 bDGlcpN 5.05 3.87 4.98 3.67 3.53 3.76-3.93
7,3,3,0,1,3,3,2,2 Ac
7,3,3,0,1,3,3,2,3 65%Ac
7,3,3,0,1,3,3,2 bDGlcpN 5.03 3.84 4.93 3.59 3.51 3.72-3.93
7,3,3,0,1,3,3,3,0,1,4 Ac
7,3,3,0,1,3,3,3,0,1 bDQuip4N 4.44 3.36 3.50 3.57 3.56 1.20
7,3,3,0,1,3,3,3,0 xDGro 3.70-3.99 4.07 3.98-4.04
7,3,3,0,1,3,3,3 P
7,3,3,0,1,3,3,4 aDGlcp 5.00 3.51 3.76 3.48 3.94 3.84-3.95
7,3,3,0,1,3,3 bDGalp 4.66 3.97 4.36 4.19 3.75 ?
7,3,3,0,1,3,2 Ac
7,3,3,0,1,3 aDGlcpN 5.05 4.03 3.95 3.65 4.20 3.78-3.85
7,3,3,0,1,4 Ac
7,3,3,0,1 bDQuip4N 4.45 3.41 3.68 3.71 3.51 1.16
7,3,3,0 xDGro 3.70-3.99 4.07 3.93-4.01
7,3,3 P
7,3 bDGalp 4.57 3.91 4.29 4.10 3.70 3.88-3.95
7,2 Ac
7 bDGlcpN 4.74 3.57 3.86 3.56 3.50 3.76-3.93
8 aXLDmanHepp 4.77 3.89 3.82 3.83 3.59 3.98 3.58-3.66
?XKdop - - 1.80-2.05 4.05 4.14 4.03 4.10 3.51-3.74
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6 C7/H7 C8/H8
7,3,2,2 Ac
7,3,2,3 65%Ac
7,3,2 bDGlcpN 99.7/5.05 53.9/3.87 76.7/4.98 67.7/3.67 76.1/3.53 60.9/3.76-3.93
7,3,3,0,1,3,3,2,2 Ac
7,3,3,0,1,3,3,2,3 65%Ac
7,3,3,0,1,3,3,2 bDGlcpN 99.7/5.03 54.0/3.84 77.2/4.93 68.0/3.59 76.1/3.51 60.9/3.72-3.93
7,3,3,0,1,3,3,3,0,1,4 Ac
7,3,3,0,1,3,3,3,0,1 bDQuip4N 102.8/4.44 73.8/3.36 73.5/3.50 56.8/3.57 71.2/3.56 16.9/1.20
7,3,3,0,1,3,3,3,0 xDGro 70.8/3.70-3.99 69.5/4.07 66.5/3.98-4.04
7,3,3,0,1,3,3,3 P
7,3,3,0,1,3,3,4 aDGlcp 100.3/5.00 72.1/3.51 72.8/3.76 69.5/3.48 72.2/3.94 60.1/3.84-3.95
7,3,3,0,1,3,3 bDGalp 100.2/4.66 74.6/3.97 77.5/4.36 76.9/4.19 75.0/3.75 60.2/?
7,3,3,0,1,3,2 Ac
7,3,3,0,1,3 aDGlcpN 97.3/5.05 52.3/4.03 79.4/3.95 67.7/3.65 71.2/4.20 60.1/3.78-3.85
7,3,3,0,1,4 Ac
7,3,3,0,1 bDQuip4N 102.8/4.45 72.5/3.41 77.3/3.68 56.7/3.71 71.2/3.51 16.5/1.16
7,3,3,0 xDGro 70.8/3.70-3.99 69.5/4.07 66.5/3.93-4.01
7,3,3 P
7,3 bDGalp 100.8/4.57 75.1/3.91 78.6/4.29 67.7/4.10 74.9/3.70 60.8/3.88-3.95
7,2 Ac
7 bDGlcpN 100.4/4.74 56.8/3.57 82.5/3.86 68.7/3.56 ?/3.50 60.8/3.76-3.93
8 aXLDmanHepp 99.3/4.77 69.9/3.89 70.8/3.82 66.2/3.83 71.4/3.59 69.3/3.98 63.7/3.58-3.66
?XKdop 34.2/1.80-2.05 65.9/4.05 66.2/4.14 69.0/4.03 75.7/4.10 64.4/3.51-3.74
report error
Found 1 record.
Displayed record 1
]; septicemia [ICD11: MA15.Y 
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