Purification and characterization of a surface protein from Lactobacillus fermentum 104R that binds to porcine small intestinal mucus and gastric mucin

Abstract

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.

FIG. 1.

Elution pattern of L. fermentum 104R protein extracts on Sephadex G-200 gel filtration chromatography. (A) SCS from strain 104R. Fractions which adhered to mucus and mucin were localized in the shaded fractions. (B) Standard curve with known proteins separated by Sephadex G-200 gel filtration chromatography in the same column. BSA, bovine serum albumin. (C) LiCl (1.0 M) extract from 104R whole cells. Fractions which adhered to mucus and mucin were localized in the shaded fractions. (D) Elution pattern after affinity chromatography using activated ch-Sepharose 4B covalently coupled to partially purified gastric mucin of a pool of adhering fractions from gel filtration chromatography of SCS eluted with different desorbing buffers, i.e., HEPES-Hanks buffer (a), 0.1 M glycine (pH 3) (b), and 0.1 M Tris (pH 8) (c). Adhesion to mucus and mucin was detected in the shaded peak.

FIG. 2.

SDS-PAGE on minigels and Western blotting of the MAPP from L. fermentum 104R, using HRP-labeled mucus for blotting. (A) Twelve percent acrylamide gels were used and stained with Coomassie blue. Lane 1, low-molecular-weight standards; lane 2, MAPP after affinity chromatography (5 μl of protein was loaded); lane 3, MAPP from native PAGE. Numbers on the left are molecular masses in kilodaltons. (B) Western blotting of the MAPP from SDS-PAGE after gel filtration chromatography of LiCl extract (lane 1), affinity chromatography of LiCl extract (lane 2), 1 M LiCl extract from 14 h culture (lane 3), and the MAPP subunit from the native MAPP band in PAGE (lane 4).

FIG. 3.

PAGE on minigels stained with Coomassie blue and Western blotting of strain 104R native MAPP. (A) PAGE (9.5%) of 1 M LiCl extract from 14-h culture; (B) Western blot of 1 M LiCl extract from 14-h culture.

FIG. 4.

Chromatograms a (top) and b (bottom) from reverse-phase HPLC of trypsin-digested peptides fractionated in a SMART system using a μRPC C2/C18 SC 2.1/10 column. Sequenced adhering peptides are marked with their fraction numbers and with arrows. The N-terminal sequence was the same as peptide 34. The sequence of peptide 5 from chromatogram a was the same as that of peptide 12 from chromatogram b. Peptide 5 from chromatogram c (not shown) was also sequenced.