Structural studies of the major glycolipids produced by two Lactobacillus johnsonii (LJ) strains, LJ 151 isolated from intestinal tract of healthy mice and LJ 142 isolated from mice with experimentally induced inflammatory bowel disease (IBD), were performed. Two major glycolipids, GL1 and GL2, were present in lipid extracts from L. johnsonii 142 and 151 strains. Glycolipid GL1 has been identified as β-D-Glcp-(1→6)-α-D-Galp-(1→2)-α-D-Glcp-diglyceride and GL2 as α-D-Galp-(1→2)-α-D-Glcp-diglyceride. The main fatty acid residues identified by gas-liquid chromatography-mass spectrometry were palmitic, stearic and lactobacillic acids. Besides structural elucidation of the major glycolipids, the aim of this study was to determine the immunochemical properties of these glycolipids and to compare their immunoreactivity to that of polysaccharides obtained from the same strains. Sera from rabbits immunized with bacterial cells possessed much higher serological reactivity with polysaccharides than with glycolipids. Inversely, reactivity of the glycolipids with human sera from patients with IBD was much higher than that determined for the polysaccharides, while reactivity of glycolipids with human sera from healthy individuals was much lower than one measured for the polysaccharides. Results indicate that glycoconjugates from Lactobacillus cell wall act as antigens and may represent new IBD diagnostic biomarkers.

structure, glycolipid, Lactobacillus johnsonii

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,2,6	bDGlcp	103.8	74.1	76.8	70.7	76.7	62.1
3,2	aDGalp	98.8	69.4	70.6	69.9	71.3	69.7
3	aDGlcp	97.5	79.3	72.6	70.0	72.5	61.8
	x?Gro	63.2	70.4	66.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,2,6	bDGlcp	4.38	3.27	3.42	3.37	3.39	3.73-3.87
3,2	aDGalp	4.96	3.80	3.78	3.92	4.32	3.85-3.98
3	aDGlcp	4.97	3.56	3.78	3.46	3.57	3.78
	x?Gro	4.19-4.42	5.24	3.65-3.83


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,2,6	bDGlcp	103.8/4.38	74.1/3.27	76.8/3.42	70.7/3.37	76.7/3.39	62.1/3.73-3.87
3,2	aDGalp	98.8/4.96	69.4/3.80	70.6/3.78	69.9/3.92	71.3/4.32	69.7/3.85-3.98
3	aDGlcp	97.5/4.97	79.3/3.56	72.6/3.78	70.0/3.46	72.5/3.57	61.8/3.78
	x?Gro	63.2/4.19-4.42	70.4/5.24	66.4/3.65-3.83

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

OCC(O)CO[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O[C@H]1O[C@H](CO[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@H](O)[C@H]1O

578.517 g/mol (C₂₁H₃₈O₁₈)

Structural studies of the major glycolipids produced by two Lactobacillus johnsonii (LJ) strains, LJ 151 isolated from intestinal tract of healthy mice and LJ 142 isolated from mice with experimentally induced inflammatory bowel disease (IBD), were performed. Two major glycolipids, GL1 and GL2, were present in lipid extracts from L. johnsonii 142 and 151 strains. Glycolipid GL1 has been identified as β-D-Glcp-(1→6)-α-D-Galp-(1→2)-α-D-Glcp-diglyceride and GL2 as α-D-Galp-(1→2)-α-D-Glcp-diglyceride. The main fatty acid residues identified by gas-liquid chromatography-mass spectrometry were palmitic, stearic and lactobacillic acids. Besides structural elucidation of the major glycolipids, the aim of this study was to determine the immunochemical properties of these glycolipids and to compare their immunoreactivity to that of polysaccharides obtained from the same strains. Sera from rabbits immunized with bacterial cells possessed much higher serological reactivity with polysaccharides than with glycolipids. Inversely, reactivity of the glycolipids with human sera from patients with IBD was much higher than that determined for the polysaccharides, while reactivity of glycolipids with human sera from healthy individuals was much lower than one measured for the polysaccharides. Results indicate that glycoconjugates from Lactobacillus cell wall act as antigens and may represent new IBD diagnostic biomarkers.

structure, glycolipid, Lactobacillus johnsonii

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,2	aDGalp	97.1	69.5	70.7	70.2	71.5	62.4
3	aDGlcp	97.0	77.2	72.3	70.4	72.4	61.8
	x?Gro	63.1	70.5	66.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,2	aDGalp	4.99	3.78	3.78	3.94	4.08	3.74-3.78
3	aDGlcp	4.98	3.56	3.76	3.42	3.55	3.69-3.80
	x?Gro	4.19-4.43	5.22	3.64-3.83


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,2	aDGalp	97.1/4.99	69.5/3.78	70.7/3.78	70.2/3.94	71.5/4.08	62.4/3.74-3.78
3	aDGlcp	97.0/4.98	77.2/3.56	72.3/3.76	70.4/3.42	72.4/3.55	61.8/3.69-3.80
	x?Gro	63.1/4.19-4.43	70.5/5.22	66.3/3.64-3.83

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

OCC(O)CO[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O[C@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O

416.376 g/mol (C₁₅H₂₈O₁₃)

The exopolysaccharide (EPS) structure from Lactobacillus johnsonii strain 151 isolated from the intestinal tract of mice was investigated. Sugar and methylation analyses together with 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, NOESY, and 1H,13C HSQC, HMBC experiments, revealed that the repeating unit of the EPS is the linear pentasaccharide: →6)-α-D-Galp-(1→6)-α-D-Glcp-(1→3)-β-D-Galf-(1→3)-α-D-Glcp-(1→2)-β-D-Galf-(1→. The immunoreactivity of two structurally different exopolysaccharides isolated from L. johnsonii, 151 and 142 (Carbohydr. Res. 2010, 345, 108-114), was compared. Both EPSs differed in their reactivity with antisera. EPS from L. johnsonii 151 reacted with anti-Lactobacillus polyclonal sera against cells of five different strains, while EPS from L. johnsonii 142 was found to react only with its own antiserum. The broader specificity and higher reactivity of EPS from 151 strain than EPS from 142 strain were also observed with human sera. The physiological antibodies recognizing polysaccharide antigens were present in both adults and umbilical cord blood sera. A highly specific EPS 142 bearing strain was isolated from experimentally induced inflammatory bowel disease (IBD) mice, while a strain with EPS 151 isolated from the intestinal tract of healthy mice is characterized by a broad immune reactivity common structure.

NMR, Exopolysaccharide structure, Lactobacillus, immunoreactivity

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@@H](O)[C@@H](CO)O[C@H](O[C@@H]2[C@@H](O)[C@H](O[C@H](CO)[C@@H]3O[C@@H](O[C@H]4[C@@H](O)[C@@H](CO)O[C@H](O[C@@H]5[C@@H](O)[C@@H](*)O[C@H](CO)[C@@H]5O)[C@@H]4O)[C@H](O)[C@H]3O)O[C@H](CO)[C@H]2O)[C@@H]1O

810.705 g/mol (C₃₀H₅₀R₂O₂₅, R = next and previous repeats, not counted in mol weight)