Carbohydrate Research 344 (2009) 2060–2062
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N-(1-Carboxyethyl)alanine (alanopine), a new non-sugar component of lipopolysaccharides of Providencia and Proteus Nina A. Kocharova, Anna N. Kondakova *, Olga G. Ovchinnikova, Andrei V. Perepelov, Alexander S. Shashkov, Yuriy A. Knirel N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation
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Article history: Received 18 June 2009 Received in revised form 9 July 2009 Accepted 13 July 2009 Available online 16 July 2009 Keywords: Providencia alcalifaciens Proteus vulgaris O-Antigen Lipopolysaccharide Alanopine 4-Amino-4,6-dideoxyglucose
a b s t r a c t O-Polysaccharides were released by mild acid degradation of lipopolysaccharides of Providencia alcalifaciens O35 and Proteus vulgaris O76 and were studied by 1D and 2D 1H and 13C NMR spectroscopies, including HMBC and NOESY (ROESY) experiments. Both polysaccharides were found to contain N-(1-carboxyethyl)alanine (alanopine) that is N-linked to 4-amino-4,6-dideoxyglucose. Analysis of published data [Vinogradov, E.; Perry, M. B. Eur. J. Biochem. 2000, 267, 2439–2446] shows that alanopine is present also on the same sugar in the lipopolysaccharide core of Proteus mirabilis O6 and O57. Ó 2009 Elsevier Ltd. All rights reserved.
Providencia and Proteus are genera of Gram-negative bacteria within the Proteeae tribe of the Enterobacteriaceae family. They are opportunistic pathogens, which under favorable conditions can cause wound and urinary tract infections and enteric diseases. Strains of both genera are highly heterogeneous in respect to O-antigens (O-polysaccharides). In most Providencia and Proteus O-serogroups, the O-polysaccharides have been analyzed chemically and a number of amino acids, such as L-alanine, L-serine, e L-threonine, D- and L-aspartic acid, L-lysine, and N -[(R)- and (S)-1-carboxyethyl]-L-lysine, have been identified as their nonsugar components (see Bacterial Carbohydrate Structure Database at http://www.glyco.ac.ru/bcsdb). Now we report on the identification of N-(1-carboxyethyl)alanine (alanopine) as a component of O-polysaccharides of Providencia alcalifaciens O35 and Proteus vulgaris O76. Lipopolysaccharides were obtained from dry bacterial cells by the phenol-water extraction and degraded with dilute acetic acid to afford high molecular mass O-polysaccharides isolated by GPC on Sephadex G-50. The O-polysaccharide from P. vulgaris O76 was O-deacetylated with aq ammonia. Sugar analysis of the O-polysaccharides by GLC of the acetylated alditols revealed, as the main components, Glc and GalN in P. alcalifaciens O35; Glc, Gal, GlcN, and fucosamine (FucN) in P. vulgaris O76; trace amounts of 4-amino-4,6-dideoxyglucose (Qui4N) in both. In addition, glucu* Corresponding author. Tel.: +7 (499) 137 6148; fax: +7 (499) 135 5328. E-mail address:
[email protected] (A.N. Kondakova). 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.07.003
ronic acid (GlcA) was identified in the former polysaccharide using a sugar analyzer. The 1H and 13C NMR spectra demonstrated pentasaccharide repeating units in both O-polysaccharides studied. Tracing connectivities in, and estimation of 3JH,H coupling constants from, the 2D 1 H, 1H COSY and TOCSY spectra combined with observation of cross-peaks between protons at nitrogen-bearing carbons and the corresponding carbons of amino sugars in the 1H, 13C-HSQC spectra revealed spin systems for Glc, GlcA, Qui4N, and two GalN residues in P. alcalifaciens O35; Glc, Gal, GlcN, FucN, and Qui4N in P. vulgaris O76. A low content of Qui4N in the polysaccharide hydrolysates could be accounted for by its significant destruction during acid hydrolysis or/and poor release from a N-acyl derivative. Each O-polysaccharide repeating unit contains three amino sugars but only two N-acetyl groups. Additional signals for two CH3CH(NH)CO groups were present in the 1D and 2D NMR spectra of both O-polysaccharides (Table 1), which could belong to two alanine residues. However, signals for both CHN carbons were shifted significantly downfield to d 57.2–58.9 as compared with their positions at d 50.1–51.8 in N-acetylalanine.1 This displacement could be due to N-alkylation, and the two groups could be parts of N-(1-carboxyethyl)alanine (alanopine, 3-aza-2,4-dimethylglutaric acid) (Chart 1). This conclusion was confirmed by C-1/ H-2, C-1/CH3-2, C-5/H-4, C-5/CH3-4, C-2/H-4, and C-4/H-2 correlations in the 1H, 13C HMBC spectra. The last two correlations, as well as C-2 and C-4 chemical shifts, excluded the possibility that the N-acyl group is an alanine dipeptide rather than alanopine.
N. A. Kocharova et al. / Carbohydrate Research 344 (2009) 2060–2062 Table 1 1 H and 13C NMR data (d, ppm) of N-(1-carboxyethyl)alanine (alanopine) in the Opolysaccharide from P. alcalifaciens O35, O-deacetylated O-polysaccharide from P. vulgaris O76, and published data3 of the core oligosaccharide from P. mirabilis O6 and O57 C-1
C-2 H-2
CH3-2 CH3-2
C-4 H-4
CH3-4 CH3-4
C-5
P. alcalifaciens O35 171.8 57.2 4.05
17.5 1.57
58.4 3.67
17.2 1.51
175.6
P. vulgaris O76 171.7 57.3 4.02
17.6 1.62
58.9 3.56
17.3 1.59
174.9
P. mirabilis O6 and O573 171.6 56.4 4.07
17 1.5
57.9 3.52
16.5 1.57
175.6
5
oligosaccharide that lacks the Qui4N derivative, the experimental and calculated molecular masses were 2282.71 and 2282.69 Da, respectively, the mass difference between the complete and incomplete cores (288.14 Da) being in agreement with an alanopine derivative of Qui4N. 1. Experimental
O HO
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1.1. Bacterial strains and growth
O H 4 N 3
CH 3
2
CH3 1
CH 3
N H
O
P. alcalifaciens O35:H18, strain 901/49 obtained from the Hungarian National Collection of Medical Bacteria (National Institute of Hygiene, Budapest) was cultivated under aerobic conditions in tryptic soy broth supplemented with 0.6% yeast extract. P. vulgaris HSC 438 (O76) isolated from a sick child at the Hospital for Sick Children, Toronto, Canada, was gifted by J. L. Penner (Centre for Diseases Control and Prevention, Atlanta, Georgia); dry bacteria were obtained from aerated liquid cultures. The bacterial mass was harvested at the end of the logarithmic growth phase, centrifuged, washed with distilled water, and lyophilized.
HO OH
OH
Chart 1. Structure of 4-[N-(1-carboxyethyl)alanyl]amino-4,6-dideoxyglucose, a component of the O-polysaccharides of P. alcalifaciens O35 and P. vulgaris O76. The absolute configurations of alanopine and Qui4N were not established; the latter is shown as D as in all other known Qui4N-containing bacterial polysaccharides.
The location of alanopine was determined by NMR spectroscopy studies of the O-polysaccharides in a 9:1 H2O/D2O mixture. This revealed signals for NH-protons, which were assigned using 2D 1H, 1 H COSY and TOCSY experiments. The 2D NOESY (ROESY) spectra showed a correlation of NH-4 proton of Qui4N with CH3-2 of alanopine at d 8.62/1.57 in the O-polysaccharide of P. alcalifaciens O35 or with H-2 of alanopine at d 8.65/4.04 in that of P. vulgaris O76. The attachment of alanopine to N-4 of Qui4N in P. alcalifaciens O35 was additionally confirmed by a correlation between C-1 of alanopine and H-4 of Qui4N at d 171.8/3.62 in the 1H, 13C HMBC spectrum. Therefore, the O-polysaccharides of P. alcalifaciens O35 and P. vulgaris O76 contain 4-[N-(1-carboxyethyl)alanyl]amino-4,6-dideoxyglucose (Chart 1). The absolute configuration of alanopine remains to be determined. Alanopine, an opine of the octopine family, is known to occur in marine invertebrates, and meso-N-(1-carboxyethyl)alanine has been isolated from squid muscle tissue.2 However, to the best of our knowledge, no alanopine has been hitherto reported as a bacterial polysaccharide component. Its discovery in Providencia and Proteus extends the list of uncommon N-acyl substituents on Qui4N, which often bears N-linked groups other than acetyl in polysaccharides of Providencia, Proteus, and some other Gram-negative bacteria (e.g., Ref. 3; see also Bacterial Carbohydrate Structure Database at http://www.glyco.ac.ru/bcsdb). 1 H and 13C NMR chemical shifts similar to those of alanopine in the O-polysaccharides of P. alcalifaciens O35 and P. vulgaris O76 have been reported for an N-acyl group on Qui4N in the lipopolysaccharide core of Proteus mirabilis O6 and O57 (Table 1), which has been identified as an alanine dipeptide.4 Our data suggest that this component has been misidentified and, in fact, is alanopine too. This conclusion was confirmed by the exact determination by high resolution ESIMS of the molecular mass 2570.85 Da for an isolated core oligosaccharide from P. mirabilis O6 (calculated molecular masses are 2570.83 and 2569.84 Da for compounds containing alanopine and alanine dipeptide, respectively; for the oligosaccharide structure see Ref. 4). For an incomplete core
1.2. Isolation of lipopolysaccharides LPS was isolated from bacterial mass of each strain by the phenol–water procedure5 followed by dialysis of the extract without layer separation. After removal of insoluble contaminations by centrifugation, the solution was freed from proteins and nucleic acids by treatment with cold (4 °C) 50% aq CCl3CO2H, the precipitate was removed by centrifugation, and the supernatant was dialyzed against distilled water and freeze-dried. 1.3. Isolation of O-polysaccharides and O-deacetylation A sample of LPS from each strain was heated with 2% acetic acid for 2.0–2.5 h at 100 °C, a lipid precipitate was removed by centrifugation, and the carbohydrate-containing supernatant was fractionated by GPC on a column (60 2.5 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer, pH 4.5, monitored using a differential refractometer (Knauer, Germany). High molecular-mass O-polysaccharides were obtained in yields 13–15% of the LPS mass. The O-polysaccharide from P. vulgaris O76 was O-deacetylated with 12% aq ammonia for 16 h at 37 °C. 1.4. Sugar analyses Polysaccharide samples were hydrolyzed with 2 M CF3CO2H (120 °C, 2 h), the monosaccharides were converted conventionally into the alditol acetates, and were analyzed by GLC on a HP-5 ms column (25 m 0.25 mm) using a Hewlett–Packard 5890 instrument (USA) and a temperature gradient of 160 °C (1 min) to 290 °C at 3 °C min 1. Uronic acids were analyzed on a LC-2000 sugar analyzer (Biotronik, Germany) as described.6 1.5. NMR spectroscopy Samples were freeze-dried twice from a 99.9% D2O soln and dissolved in 99.95% D2O. 1H and 13C NMR spectra were recorded at 30 °C on a Bruker AV600 spectrometer (Germany) using internal TSP (dH 0) and acetone (dC 31.45) as references. 2D NMR spectra were obtained using standard Bruker software, and Bruker TOPSPIN 2.1 program was employed to acquire and process the NMR data. A mixing time of 200 and 150 ms was used in NOESY and TOCSY experiments, respectively. Other NMR experimental parameters were set essentially as described.7
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1.6. Mass spectrometry Electrospray ionization Fourier transform ion-cyclotron resonance MS was performed in the negative ion mode using an APEXII instrument (Bruker Daltonics, USA) equipped with a 7 Tesla magnet and an Apollo ion source. Mass spectra were acquired using standard experimental sequences as provided by the manufacturer. The mass scale was calibrated externally using compounds of known structure. Samples (10 ng ll 1) were dissolved in a 50:50:0.001 (v/v/v) mixture of 2-propanol/water/triethylamine, pH 8.5, and sprayed at a flow rate of 2 lL min 1. The capillary entrance and exit voltage was set to 3.8 kV and 100 V, respectively, and drying gas temperature was set to 150 °C. Acknowledgments Authors thank Prof. Z. Sidorczyk and Prof. A. Rozalski for providing LPS samples of Proteus and Providencia and Dr. B. Lindner (Re-
search Centre Borstel, Germany) for providing access to the mass spectrometer. This work was supported by the Russian Foundation for Basic Research (Grant 08-04-92221). References 1. Sidorczyk, Z.; Swierzko, A.; Vinogradov, E. V.; Knirel, Y. A.; Shashkov, A. S. Arch. Immunol. Ther. Exp. 1994, 42, 209–215. 2. Thompson, J.; Donkersloot, J. A. Annu. Rev. Biochem. 1992, 61, 517–557. 3. Kondakova, A. N.; Lindner, B.; Fudala, R.; Senchenkova, S. N.; Moll, H.; Shashkov, A. S.; Kaca, W.; Zähringer, U.; Knirel, Y. A. Biochemistry (Moscow) 2004, 69, 1034– 1043. 4. Vinogradov, E.; Perry, M. B. Eur. J. Biochem. 2000, 267, 2439–2446. 5. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91. 6. Shashkov, A. S.; Toukach, F. V.; Senchenkova, S. N.; Ziolkowski, A.; Paramonov, N. A.; Kaca, W.; Knirel, Y. A.; Kochetkov, N. K. Biochemistry (Moscow) 1997, 62, 509– 513. 7. Hanniffy, O.; Shashkov, A. S.; Senchenkova, S. N.; Tomshich, S. V.; Komandrova, N. A.; Romanenko, L. A.; Knirel, Y. A.; Savage, A. V. Carbohydr. Res. 1999, 321, 132–138.
