Hostname: page-component-7bb8b95d7b-qxsvm Total loading time: 0 Render date: 2024-09-17T11:57:09.473Z Has data issue: false hasContentIssue false
  • English
  • Français

Detection and localization of peptidases in Lactococcus lactis with monoclonal antibodies

Published online by Cambridge University Press:  01 June 2009

Harry Laan ,
Roel E. Haverkort ,
Loe De Leij  and
Wil N. Konings
Harry Laan
Affiliation:
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN HarenThe Netherlands
Roel E. Haverkort
Affiliation:
Department of Clinical Immunology, University of Groningen, Oostersingel 59, NL-9713 EZ Groningen, The Netherlands
Loe De Leij
Affiliation:
Department of Clinical Immunology, University of Groningen, Oostersingel 59, NL-9713 EZ Groningen, The Netherlands
Wil N. Konings
Affiliation:
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN HarenThe Netherlands
Get access Rights & Permissions [Opens in a new window]

Summary

Monoclonal antibodies against peptidases of Lactococcus lactis were isolated and characterized: PEPN1–4 against a lysyl aminopeptidase PepN, PEPT1–5 against a tripeptidase PepT and PEPD1–3 against a dipeptidase PepD. These monoclonal antibodies reacted specifically with their respective antigens in crude cell extracts of Lc. lactis subspp. cremoris and lactis. A number of monoclonal antibodies cross reacted with proteins of other (lactic acid) bacteria. PEPT1, 2, 4 and 5 cross reacted weakly with a 35 kDa protein in Lactobacillus delbrueckii, while PEPT1 and PEPT2 reacted with proteins in the cell-free extract of Streptococcus thermophilus and Clostridium fervidus. Of the four isolated monoclonal antibodies against PepN, only PEPN3 cross reacted weakly with a 90 kDa protein in Escherichia coli cell-free extract, and the other three antibody species against PepN cross reacted with 80 kDa proteins of Lb. casei, Lb. delbrueckii, and Str. bovis, but not of Esch. coli. Of the three monoclonal antibodies against PepD, only PEPD1 and PEPD2 cross reacted with 40 kDa proteins of Lb. casei, Lb. delbrueckii and Str. bovis. All PEPN, PEPD and PEPT antibodies reacted with components in cell-free extracts of eleven different Lc. lactis strains, indicating that the peptidases of these strains were very similar to those of Lc. lactis subsp. cremoris WG2. However, Lc. lactis subsp. hordniae appeared to differ from the other Lc. lactis subspecies since only PEPT1, 2 and 5 reacted with a protein in the cell-free extract. Immunogold labelling of Lc. lactis WG2 with the isolated monoclonal antibodies revealed that PepN, PepD and PepT were located intracellularly. The intracellular location of these peptidases is discussed in relation to the supply of essential amino acids and peptides.

Type
Original Articles
Information
Journal of Dairy Research , Volume 63 , Issue 2 , May 1996 , pp. 245 - 256
Copyright
Copyright © Proprietors of Journal of Dairy Research 1996

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Bosman, B. W., Tan, P. S. T. & Konings, W. N. 1990 Purification and characterization of a tripeptidase from Lc. lactis subsp. cremoris WG2. Applied and Environmental Microbiology 56 1839–1843CrossRefGoogle Scholar
Chapot-Chartier, M. -P., Nardi, M., Chopin, M. -C., Chopin, A. & Gripon, J. -C. 1993 Cloning and sequencing of pepC, a cysteine aminopeptidase gene from Lc. lactis subsp. cremoris AM2. Applied and Environmental Microbiology 59 330–333CrossRefGoogle Scholar
De Leij, L., Poppema, S. & The, T. H. 1983 Cryopreservation of newly formed hybridomas. Journal of Immunological Methods 62 69–72CrossRefGoogle ScholarPubMed
De Man, J. C., Rogosa, M. & Sharpe, M. E. 1960 A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23 130–135CrossRefGoogle Scholar
Eggiman, B. & Bachmann, M. 1980 Purification and partial characterization of an aminopeptidase from Lactobacillus lactis. Applied and Environmental Microbiology 40 876–882CrossRefGoogle Scholar
Hagting, A., Kunji, E. R. S., Leenhouts, K. J., Poolman, B. & Konings, W. N. 1994 The dipeptide and tripeptide transport protein of Lactococcus lactis. A new type of bacterial peptide transporter. Journal of Biological Chemistry 269 11391–11399CrossRefGoogle ScholarPubMed
Harlow, E. & Lane, D. P. 1988 Antibodies: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor LaboratoryGoogle Scholar
Juillard, V., Laan, H., Kunji, E. R. S., Jeronimus-Stratingh, C. M., Bruins, A. P. and Konings, W. N. 1995 a The extracellular P1-type proteinase of Lactococcus lactis hydrolyzes β-casein into more than one hundred different oligopeptides. Journal of Bacteriology 177, 3472–3478CrossRefGoogle Scholar
Juillard, V., Le Bars, D., Kunji, E. R. S., Konings, W. N., Gripon, J. -C. & Richard, J. 1995 b Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk. Applied and Environmental Microbiology 61 3024–3030CrossRefGoogle ScholarPubMed
Kennett, R. H. 1980 Fusion protocols, fusion by centrifugation of cells suspended in polyethylene glycol. In Monoclonal Antibodies, pp. 365–367 (Eds Kennett, R. H., McKarn, T. J. and Bechtil, K. B.). New York: PlenumCrossRefGoogle Scholar
Konings, W. N., Poolman, B. & Driessen, A. J. M. 1989 Bioenergetics and solute transport in lactococui. Critical Reviews in Microbiology 16 419–476CrossRefGoogle Scholar
Kunji, E. R. S., Hagting, A., de Vries, C. J., Juillard, V., Haandrikman, A. J., Poolman, B. & Konings, W. N. 1995 Transport of β-casein-derived peptides by the oligopeptide transport system is a crucial step in the protcolytic pathway of Lactococcus lactis. Journal of Biological Chemistry 270 1569–1574CrossRefGoogle ScholarPubMed
Kunji, E. R. S., Smid, E. J., Plapp, R., Poolman, B. & Konings, W. N. 1993 Di/tri- and oligopeptides are taken up via distinct transport mechanisms in Lc. lactis. Journal of Bacteriology 175 2052–2059CrossRefGoogle Scholar
Kyhse-Andersen, J. 1984 Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. Journal of Biochemical and Biophysical Methods 10 203–209CrossRefGoogle ScholarPubMed
Laan, J., Kok, J., Haandrikman, A. J., Venema, G. & Konings, W. N. 1992 Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis. European Journal of Biochemistry 204 815–820CrossRefGoogle ScholarPubMed
Laan, H., Smid, E. J., de Leij, L., Schwander, E. & Konings, W. N. 1988 Monoclonal antibodies to the cell wall associated proteinase of Lc. lactis subsp. cremoris WG2. Applied and Environmental Microbiology 54 2250–2256CrossRefGoogle Scholar
Laan, H., Smid, E. J., Tan, P. S. T. & Konings, W. N. 1989 Enzymes involved in the degradation and utilization of casein in Lc. lactis. Netherlands Milk and Dairy Journal 43 327–345Google Scholar
Lowry, O. H., Rosebrough, N. J., Farr, A. J. & Randall, R. J. 1951 Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193 265–275CrossRefGoogle ScholarPubMed
Mayo, B., Kok, J., Venema, K., Bockelmann, W., Teuber, M., Reinke, H. & Venema, G. 1991 Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lc. lactis subsp. cremoris. Applied and Environmental Microbiology 57 38–44CrossRefGoogle Scholar
Mierau, I., Haandrikman, A. J., Velterop, O., Tan, P. S. T., Leenhouts, K. L., Konings, W. N., Venema, G. & Kok, J. 1994 Tripeptidase gene (pepT) of Lactococcus lactis: molecular cloning and nuclcotide sequencing of pepT and construction of a chromosomal deletion mutant. Journal of Bacteriology 176, 2854–2861.CrossRefGoogle Scholar
Mierau, I., Tan, P. S. T., Haandrikman, A. J., Mayo, B., Kok, J., Leenhouts, K. L., Konings, W. N. & Venema, G. 1993 Cloning and sequencing of the gene for a lactococcal endopeptidasc, an enzyme with sequence similarity to mammalian enkephalinase. Journal of Bacteriology 175 2087–2096CrossRefGoogle ScholarPubMed
Nardi, M., Chopin, M. -C., Chopin, A., Cals, M. -M. & Gripon, J. -C. 1991 Cloning and DNA sequence analysis of an X-propyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. lactis NCDO763. Applied and Environmental Microbiology 57 45–50CrossRefGoogle Scholar
Schleifer, K. H., Kraus, J., Dvorak, C., Kilppek-Bälz, R., Collins, M. D. & Fischer, W. 1985 Transfer of Streptococcus lactis and related streptococci to the genus Lactococcus gen. nov. Systematic and Applied Microbiology 6 183–195CrossRefGoogle Scholar
Slot, J. W. & Geuze, H. J. 1984 Gold markers for single and double immunolabelling of ultrathin cryosections. In Immunolabelling for Electron Microscopy, pp. 129–142 (Eds Polak, J. M. and Varndell, I. M.). New York: Elsevier Science PublishingGoogle Scholar
Smid, E. J., Driessen, A. J. M. & Konings, W. N. 1989 Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis. Journal of Bacteriology 171 292–298CrossRefGoogle ScholarPubMed
Strøman, P. 1992 Sequence of a gene (lap) encoding a 953-kDa aminopeptidase from Lactococcus lactis subsp. cremoris WG2. Gene 113 107–112CrossRefGoogle Scholar
Tan, P. S. T., Chapot-Chartier, M. -P., Pos, K. M., Rousseau, M., Boquien, C. -Y., Gripon, J. -C. & Konings, W. N. 1992 a Localization of peptidases in lactococci. Applied and Environmental Microbiology 58 285–290CrossRefGoogle ScholarPubMed
Tan, P. S. T. & Konings, W. N. 1990 Purification and characterization of an aminopeptidase from Lc. lactis subsp. cremoris WG2. Applied and Environmental Microbiology 56 526–532CrossRefGoogle Scholar
Tan, P. S. T., Poolman, B. & Konings, W. N. 1993 Review article. Proteolytic enzymes of Lactococcus lactis. Journal of Dairy Research 60 269–286CrossRefGoogle ScholarPubMed
Tan, P. S. T., Van Alen-Boerrigter, I. J., Poolman, B., Siezen, R. J., de Vos, W. M. & Konings, W. N. 1992 b Characterization of the Lc. lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N. FEBS Letters 306 9–16CrossRefGoogle Scholar
Van Alen-Boerrigter, I. J., Baankreis, R. & de Vos, W. M. 1991 Characterization and overexpression of the Lc. lactis pepN gene and localization of its product, aminopeptidase-N. Applied and Environmental Microbiology 57 2555–2561CrossRefGoogle Scholar
Van Boven, A., Tan, P. S. T. & Konings, W. N. 1988 Purification and characterization of a dipeptidase from Streptococcus cremoris WG2. Applied and Environmental Microbiology 54 43–49CrossRefGoogle ScholarPubMed