β-Aspartyl-ε-lysine, a peptide of the fecal contents of axenic mice

β-Aspartyl-ϵ-lysine, a peptide of the fecal contents of axenic mice J.-P. PÉLISSIER, Françoise DUBOS Laboratoire de Biochimie et Techno%gie laitières, ) Laboratoire d’Ecologie Microbienne, * ( I.N.R.A., 78350Jouy-en-Josas, France. Summary. Small quantities of low-molecular weight peptides have been characterized in the feces of axenic mice. In fecal material of axenic mice fed an autoclaved synthetic (SN) diet, we isolated a dipeptide and characterized its structure as f3-aspartyl-¡;-lysine. This product was also present in the feces of gnotobiotic mice harbouring Clostridium perenne. We could not detect the product in the fecal contents of holoxenic mice, Clostridium diffici/e-contaminated mice or axenic mice fed the irradiated SN diet. The peptide, fl-aspartyl-lysine, was produced by heating proteins during sterilization, E causing the formation of a pseudopeptide bond between the e-amino group of lysine and the amide group of asparagine. The intestinal strains seemed to differ in their ability to split this pseudopeptide bond in vivo. Introduction. The fecal microbial flora of newborn mammals seems to depend largely on their diet, which varies between birth and weaning (Decuypere and Van Der Heyde, 1972 ; Chopin et al., 1974 ; Ducluzeau et al., 1974 ; Savage, 1977). As shown by Ducluzeau et al. (1981), a strain of Clostridium perenne did not establish when axenic mice were fed with a « non-permissive» diet, but it did establish when they were fed a « permissive» diet. Once this strain had been established in animals fed a permissive diet, it persisted in the dominant flora, even if the permissive diet was replaced by a non-permissive one (Ducluzeau et al., 19811.). The supernatant of fecal and cecal contents of mice contains free amino acids. Higher amounts of free amino acid have been found in the cecal contents of axenics than in those of conventional mice (Combe et al., 1965). Only one low-molecular weight peptide substance, (3-aspartylglycine has been characterized in the cecal contents of axenic mice (Welling and Groen, 1978 ; Welling et a/., 1980). The fecal or cecal digestive content of axenic animals fed with the nonpermissive diet in vivo inhibits the growth of C. perenne (Dubos, 1982). The present paper describes the isolation and characterization of a low-molecular weight peptide in the inhibitory fraction of the fecal contents of axenic mice fed a non- permissive diet. We have also tried to determine if the presence of this peptide in the gut depended on the bacterial strain harboured. The effect of this peptide on the growth of C. perenne has been studied by Dubos (1982). Experimental protocol Products. ― a-L-aspartylglycine, ¡3-L-aspartylglycine and a-L-aspartyl-¡;-Llysine were from Bachem (Budendorf, Switzerland) ; dansyl chloride was from Fluka AG (Buchs, Switzerland) ; Sephadex G25 was from Pharmacia (Uppsala, Sweden) ; Whatman 3MM chromatography paper was from Whatman (Maidstone, Kent, England) ; ninhydrin was from Merck (Darmstadt, West Germany) and 2 4,6 collidin was from Sigma (St Louis, Mo., USA). All the other products ; were from Prolabo (Paris, France). , Animals. ― Adult axenic C3H mice were reared in plastic Trexler-type isolators. Gnotobiotic mice harbouring Clostridium strains were reared in the same type of isolators. Diets. ― Axenic mice were fed ad libitum a semi-synthetic (SN) diet containing (in g/kg) : 220 g of casein, 580 g of cornstarch, 90 g of corn oil, 50 g of cellulose and 45 g of a mineral and vitamin mixture ; this diet was sterilized by autoclaving at 120 °C for 20 min. The same diet without casein was used as a protein-free (PF) diet. The SN diet and a commercial one (UAR n R03) sterilized by !y-irradiation (4 M rad) were also given. Bacterial strains. A strain of C. perenne and one of Clostridium difficile from the collection of the « Laboratoire d’Ecologie Microbienne» were cultured on a standard liquid medium for anaerobes. Axenic mice were inoculated per os with a 24-hour culture of C. difficile or C. perenne. - Isolation of low-mo%cular weight substances. ― Batches of fecal contents prepared by pooling 20 g of the fresh feces of 10 axenic mice. The products were mixed with 40 ml of water and homogenized in an Ultraturrax (Garantlab-Turrax, Paris, France). The supernatants were obtained by centrifugation at 20 000 x g for 10 min and then stored at - 25 °C. 100 ml of the supernatant were applied to a Sephadex G25 (fine) column (3 x 180 cm) and eluted at a flow rate of 20 ml/h with 30 % (v/v) acetic acid. 10-ml fractions were collected. 100 pl of the fractions were spotted on Whatman 3MM paper and subjected to high-voltage paper electrophoresis at pH 1.9 (formic acid/acetic acid/water, 25:87:888, v/v/v) for 30 min at 53 V/cm. A reference mixture (Beckman, Palo Alto, USA) containing all the common amino acids were used. After drying at 55 °C, the paper was coloured with a ninhydrin-collidine solution (ninhydrin/ collidine/ethanol ; 1 g, 50 ml, 950 ml) and heated at 55 °C for 5 min. Preparative high-voltage electrophoresis was carried out at pH 1.9. The products were eluted from paper with 30 % acetic acid. were ldentification of the peptide substance. ― Amino acid analysis was performed with a Multichrom amino acid analyzer (Beckman, Munchen, West Germany). The samples were hydrolyzed in 1.5 ml of 5.7 N HCI at 110 °C in evacuated, sealed glass-tubes for 24 h. N-terminal amino acid was determined as described by Hartley (1970). Mass spectrometry was obtained on a Nermag R10R10. Results Fractions of axenic mice fed the autoclaved SN diet were obtained by Sephadex chromatography. Using high-voltage paper electrophoresis, we detected, in the inhibitory fraction, a ninhydrin reactive product migrating between free alanine and free glycine (fig. 1). This product was eluted from the Sephadex G25 just before the major part of the free amino acids. When the same purifications were carried out on the fecal contents of gnotobiotic mice harbouring C. perenne and fed the autoclaved SN diet, the same spot in about equal amounts was observed. The product was not seen (fig. 2) in fractions of conventional mice or gnotobiotic mice harbouring C. difficile and fed the autoclaved SN diet. We did not find the peptide with the PF diet. With the irradiated SN diet, obtained significantly lower quantities of the peptide than with the same diet sterilized by heat. We did not find significant quantities of the peptide in the diets themselves analyzed by the same method. After acid hydrolysis, the substance purified by paper electrophoresis gave equal amounts of aspartic acid and lysine by amino acid analysis. The molecular weight obtained by mass spectrometry was that of a dipeptide composed of one residue of aspartic acid and one residue of lysine. Dansylation and subsequent hydrolysis showed the N-terminal amino acid to be aspartic acid. Under high-voltage paper electrophoresis, comm (...truncated)