E.K.H. Schweda <elksc
Clinical Research Centre, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden
A heterogeneous population of glycoforms expressed by NTHi strains 1158 and 1159 has been elucidated using NMR spectroscopy and capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) materials, as well as HPLC-ESI-MS(n) on dephosphorylated and methylated OS samples. The most abundant glycoform contained a disaccharide chain: PCho→7)-D-α-D-Hepp-(1→6)-β-D-Glcp linked to HepI from the common structural element of H. influenzae LPS: L-α-D-HepIIIp-(1→2)-[PEtn→6]-L-α-D-HepIIp-(1→3)-L-α-D-HepIp-(1→5)-[PPEtn→4]-α-Kdop-(2→6)-lipid A. Phosphocholine (PCho) was found at two positions in the LPS glycoforms; PCho substituted the 6-position of β-D-Glcp attached to HepIII and was also located at a novel position linked to D-α-D-Hepp; this latter position was determined by structural analysis of LPS from a 1158lpsA mutant strain. Additionally, HPLC-ESI-MS(n) experiments indicated glycoforms that have chain elongation from HepII, this was found only in glycoforms, which lack the additional heptose in the outer core region. Structural details of these glycoforms were confirmed by analyses of LPS from a 1158losB2 mutant strain; the losB2 gene is required for addition of the D,D-Hep to the outer core region in strain 1158.
13C NMR, 1H NMR, methylation, NMR-2D, GC-MS, HF solvolysis, sugar analysis, 31P NMR, ESI-MS, mild acid hydrolysis, de-O-acylation with hydrazine, NMR-1D, HPLC, CE-ESI-MS
NMR chemical shift data for O-deacylated OS from NTHi 1158. Published erroneous NMR chemical shift of #5,3,3,4,4,4_aDGalp С3 (72.7) was removed by CSDB staff
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7
5,3,2,2,6,0 xXCho 59.8 66.2 ?
5,3,2,2,6 P
5,3,2,2 bDGlcp 102.7 73.1 75.5 68.7 74.5 64.5
5,3,2 aXLDmanHepp 99.5 79.2 68.1 66.3 ? ? ?
5,3,3,4,4,4,3,2 Ac
5,3,3,4,4,4,3 bDGalpN 102.5 52.6 ? ? ? ?
5,3,3,4,4,4 aDGalp 100.8 71.0 ? 68.8 ? ?
5,3,3,4,4 bDGalp 102.3 72.0 71.9 77.4 75.7 ?
5,3,3,4 bDGlcp 102.7 73.1 75.1 78.5 75.0 60.6
5,3,3 aDGlcp 100.1 71.9 71.4 78.7 71.0 60.8
5,3,6,0 xXEtN 62.1 40.2
5,3,6 P
5,3 aXLDmanHepp 98.6 78.5 77.4 ? 72.4 74.7 63.7
5,4 bDGlcp 104.0 73.2 76.8 70.2 76.8 61.0
5 aXLDmanHepp 96.7 69.9 72.5 73.5 ? 68.0 ?
4,0,0 xXEtN
4,0 P
4 P
aXKdop
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7
5,3,2,2,6,0 xXCho 4.35 3.70 ?
5,3,2,2,6 P
5,3,2,2 bDGlcp 4.44 3.37 3.54 3.58 3.58 4.17
5,3,2 aXLDmanHepp 5.12 4.00 3.96 3.76 ? ? ?
5,3,3,4,4,4,3,2 Ac
5,3,3,4,4,4,3 bDGalpN 4.64 3.96 3.76 3.96 ? ?
5,3,3,4,4,4 aDGalp 4.93 3.91 3.97 4.27 ? ?
5,3,3,4,4 bDGalp 4.53 3.60 3.76 4.05 3.80 ?
5,3,3,4 bDGlcp 4.57 3.40 3.70 3.70 3.66 3.85-4.01
5,3,3 aDGlcp 5.33 3.62 3.87 3.71 3.91 3.96
5,3,6,0 xXEtN 4.15 3.29
5,3,6 P
5,3 aXLDmanHepp 5.69 4.29 4.11 ? 3.73 4.59 3.90
5,4 bDGlcp 4.53 3.36 3.46 3.42 3.46 3.78-3.97
5 aXLDmanHepp 5.05 4.00 4.05 4.29 ? 4.15 ?
4,0,0 xXEtN
4,0 P
4 P
aXKdop
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6 C7/H7
5,3,2,2,6,0 xXCho 59.8/4.35 66.2/3.70 ?/?
5,3,2,2,6 P
5,3,2,2 bDGlcp 102.7/4.44 73.1/3.37 75.5/3.54 68.7/3.58 74.5/3.58 64.5/4.17
5,3,2 aXLDmanHepp 99.5/5.12 79.2/4.00 68.1/3.96 66.3/3.76 ?/? ?/? ?/?
5,3,3,4,4,4,3,2 Ac
5,3,3,4,4,4,3 bDGalpN 102.5/4.64 52.6/3.96 ?/3.76 ?/3.96 ?/? ?/?
5,3,3,4,4,4 aDGalp 100.8/4.93 71.0/3.91 ?/3.97 68.8/4.27 ?/? ?/?
5,3,3,4,4 bDGalp 102.3/4.53 72.0/3.60 71.9/3.76 77.4/4.05 75.7/3.80 ?/?
5,3,3,4 bDGlcp 102.7/4.57 73.1/3.40 75.1/3.70 78.5/3.70 75.0/3.66 60.6/3.85-4.01
5,3,3 aDGlcp 100.1/5.33 71.9/3.62 71.4/3.87 78.7/3.71 71.0/3.91 60.8/3.96
5,3,6,0 xXEtN 62.1/4.15 40.2/3.29
5,3,6 P
5,3 aXLDmanHepp 98.6/5.69 78.5/4.29 77.4/4.11 ?/? 72.4/3.73 74.7/4.59 63.7/3.90
5,4 bDGlcp 104.0/4.53 73.2/3.36 76.8/3.46 70.2/3.42 76.8/3.46 61.0/3.78-3.97
5 aXLDmanHepp 96.7/5.05 69.9/4.00 72.5/4.05 73.5/4.29 ?/? 68.0/4.15 ?/?
4,0,0 xXEtN
4,0 P
4 P
aXKdop
report error
Found 1 record.
Displayed record 1
, Life stage: adult]; sinusitis [ICD11: SC91 
ifm.liu.se>