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. 1997 Oct 1;327(Pt 1):95–103. doi: 10.1042/bj3270095

Functional diversity and interactions between the repeat domains of rat intestinal lactase.

B Jost 1, I Duluc 1, M Richardson 1, R Lathe 1, J N Freund 1
PMCID: PMC1218768  PMID: 9355740

Abstract

Lactase-phlorizin hydrolase (LPH), a major digestive enzyme in the small intestine of newborns, is synthesized as a high-molecular-mass precursor comprising four tandemly repeated domains. Proteolytic cleavage of the precursor liberates the pro segment (LPHalpha) corresponding to domains I and II and devoid of known enzymic function. The mature enzyme (LPHbeta) comprises domains III and IV and is anchored in the brush border membrane via a C-terminal hydrophobic segment. To analyse the roles of the different domains of LPHalpha and LPHbeta, and the interactions between them, we have engineered a series of modified derivatives of the rat LPH precursor. These were expressed in cultured cells under the control of a cytomegalovirus promoter. The results show that recombinant LPHbeta harbouring both domains III and IV produces lactase activity. Neither domain III nor IV is alone sufficient to generate active enzyme, although the corresponding proteins are transport-competent. Tandem duplication of domains III or IV did not restore lactase activity, demonstrating the separate roles of both domains within LPHbeta. Further, the development of lactase activity did not require LPHalpha; however, LPHalpha potentiated the production of active LPHbeta but the individual LPHalpha subdomains I and II were unable to do so. Lactase activity and targeting required the C-terminal transmembrane anchor of LPH; this requirement was terminal transmembrane anchor or LPH; this requirement was not satisfied by the signal/anchor region of another digestive enzyme: sucrase-isomaltase. On the basis of this study we suggest that multiple levels of intramolecular interactions occur within the LPH precursor to produce the mature enzyme, and that the repeat domains of the precursor have distinct and specific functions in protein processing, substrate recognition and catalysis. We propose a functional model of LPHbeta in which substrate is channelled from an entry point located within domain II to the active site located in domain IV.

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Selected References

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  1. Birkenmeier E., Alpers D. H. Enzymatic properties of rat lactase-phlorizin hydrolase. Biochim Biophys Acta. 1974 May 20;350(1):100–112. doi: 10.1016/0005-2744(74)90207-1. [DOI] [PubMed] [Google Scholar]
  2. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  3. Boukamel R., Freund J. N. The rat LPH gene 5' region: comparative structure with the human gene. DNA Seq. 1992;3(2):119–121. doi: 10.3109/10425179209034006. [DOI] [PubMed] [Google Scholar]
  4. Büller H. A., Montgomery R. K., Sasak W. V., Grand R. J. Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat. J Biol Chem. 1987 Dec 15;262(35):17206–17211. [PubMed] [Google Scholar]
  5. Chantret I., Lacasa M., Chevalier G., Ruf J., Islam I., Mantei N., Edwards Y., Swallow D., Rousset M. Sequence of the complete cDNA and the 5' structure of the human sucrase-isomaltase gene. Possible homology with a yeast glucoamylase. Biochem J. 1992 Aug 1;285(Pt 3):915–923. doi: 10.1042/bj2850915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colombo V., Lorenz-Meyer H., Semenza G. Small intestinal phlorizin hydrolase: the "beta-glycosidase complex". Biochim Biophys Acta. 1973 Dec 19;327(2):412–424. doi: 10.1016/0005-2744(73)90425-7. [DOI] [PubMed] [Google Scholar]
  7. Danielsen E. M. Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase. Biochemistry. 1990 Jan 9;29(1):305–308. doi: 10.1021/bi00453a042. [DOI] [PubMed] [Google Scholar]
  8. Danielsen E. M. Dimeric assembly of enterocyte brush border enzymes. Biochemistry. 1994 Feb 15;33(6):1599–1605. doi: 10.1021/bi00172a041. [DOI] [PubMed] [Google Scholar]
  9. Dudley M. A., Hachey D. L., Quaroni A., Hutchens T. W., Nichols B. L., Rosenberger J., Perkinson J. S., Cook G., Reeds P. J. In vivo sucrase-isomaltase and lactase-phlorizin hydrolase turnover in the fed adult rat. J Biol Chem. 1993 Jun 25;268(18):13609–13616. [PubMed] [Google Scholar]
  10. Duluc I., Boukamel R., Mantei N., Semenza G., Raul F., Freund J. N. Sequence of the precursor of intestinal lactase-phlorizin hydrolase from fetal rat. Gene. 1991 Jul 22;103(2):275–276. doi: 10.1016/0378-1119(91)90286-k. [DOI] [PubMed] [Google Scholar]
  11. Duluc I., Freund J. N., Leberquier C., Kedinger M. Fetal endoderm primarily holds the temporal and positional information required for mammalian intestinal development. J Cell Biol. 1994 Jul;126(1):211–221. doi: 10.1083/jcb.126.1.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Duluc I., Freund J. N., Mantei N., Raul F. L'ADNc de la lactase détecte un ARNm qui s'exprime différemment dans le jéjunum et le côlon au cours du développement du rat. Gastroenterol Clin Biol. 1990;14(8-9):635–640. [PubMed] [Google Scholar]
  13. Duluc I., Jost B., Freund J. N. Multiple levels of control of the stage- and region-specific expression of rat intestinal lactase. J Cell Biol. 1993 Dec;123(6 Pt 1):1577–1586. doi: 10.1083/jcb.123.6.1577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Freund J. N. Do co-translated product(s) of lactase-phlorizin hydrolase accumulate in the rat intestine? Cell Mol Biol (Noisy-le-grand) 1992 Nov;38(7):713–718. [PubMed] [Google Scholar]
  15. Freund J. N., Gossé F., Raul F. Derivatives of plant beta-glucans are hydrolyzed by intestinal lactase-phlorizin hydrolase of mammals. Enzyme. 1991;45(1-2):71–74. doi: 10.1159/000468868. [DOI] [PubMed] [Google Scholar]
  16. Freund J. N., Jost B., Lorentz O., Duluc I. Identification of homologues of the mammalian intestinal lactase gene in non-mammals (birds and molluscs). Biochem J. 1997 Mar 1;322(Pt 2):491–498. doi: 10.1042/bj3220491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gräbnitz F., Seiss M., Rücknagel K. P., Staudenbauer W. L. Structure of the beta-glucosidase gene bglA of Clostridium thermocellum. Sequence analysis reveals a superfamily of cellulases and beta-glycosidases including human lactase/phlorizin hydrolase. Eur J Biochem. 1991 Sep 1;200(2):301–309. doi: 10.1111/j.1432-1033.1991.tb16186.x. [DOI] [PubMed] [Google Scholar]
  18. Gus-Mayer S., Brunner H., Schneider-Poetsch H. A., Rüdiger W. Avenacosidase from oat: purification, sequence analysis and biochemical characterization of a new member of the BGA family of beta-glucosidases. Plant Mol Biol. 1994 Nov;26(3):909–921. doi: 10.1007/BF00028858. [DOI] [PubMed] [Google Scholar]
  19. Hauri H. P., Kedinger M., Haffen K., Freiburghaus A., Grenier J. F., Hadorn B. Biosynthesis of brush border glycoproteins by human small intestinal mucosa in organ culture. Biochim Biophys Acta. 1977 Jun 16;467(3):327–339. doi: 10.1016/0005-2736(77)90310-8. [DOI] [PubMed] [Google Scholar]
  20. Hawkins A. R., Lamb H. K. The molecular biology of multidomain proteins. Selected examples. Eur J Biochem. 1995 Aug 15;232(1):7–18. doi: 10.1111/j.1432-1033.1995.tb20775.x. [DOI] [PubMed] [Google Scholar]
  21. Hays W. S., Jenison S. A., Yamada T., Pastuszyn A., Glew R. H. Primary structure of the cytosolic beta-glucosidase of guinea pig liver. Biochem J. 1996 Nov 1;319(Pt 3):829–837. doi: 10.1042/bj3190829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1991 Dec 1;280(Pt 2):309–316. doi: 10.1042/bj2800309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hunziker W., Spiess M., Semenza G., Lodish H. F. The sucrase-isomaltase complex: primary structure, membrane-orientation, and evolution of a stalked, intrinsic brush border protein. Cell. 1986 Jul 18;46(2):227–234. doi: 10.1016/0092-8674(86)90739-7. [DOI] [PubMed] [Google Scholar]
  24. Jacob R., Bulleid N. J., Naim H. Y. Folding of human intestinal lactase-phlorizin hydrolase. J Biol Chem. 1995 Aug 4;270(31):18678–18684. doi: 10.1074/jbc.270.31.18678. [DOI] [PubMed] [Google Scholar]
  25. Jacob R., Radebach I., Wüthrich M., Grünberg J., Sterchi E. E., Naim H. Y. Maturation of human intestinal lactase-phlorizin hydrolase: generation of the brush border form of the enzyme involves at least two proteolytic cleavage steps. Eur J Biochem. 1996 Mar 15;236(3):789–795. doi: 10.1111/j.1432-1033.1996.t01-1-00789.x. [DOI] [PubMed] [Google Scholar]
  26. Jacob R., Zimmer K. P., Naim H., Naim H. Y. The apical sorting of lactase-phlorizin hydrolase implicates sorting sequences found in the mature domain. Eur J Cell Biol. 1997 Jan;72(1):54–60. [PubMed] [Google Scholar]
  27. Keller P., Semenza G., Shaltiel S. Disposition of the carboxy-terminus tail of rabbit lactase-phlorizin hydrolase elucidated by phosphorylation with protein kinase A in vitro and in tissue culture. FEBS Lett. 1995 Jul 24;368(3):563–567. doi: 10.1016/0014-5793(95)00679-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Keller P., Zecca L., Boukamel R., Zwicker E., Gloor S., Semenza G. Furin, PC1/3, and/or PC6A process rabbit, but not human, pro-lactase-phlorizin hydrolase to the 180-kDa intermediate. J Biol Chem. 1995 Oct 27;270(43):25722–25728. doi: 10.1074/jbc.270.43.25722. [DOI] [PubMed] [Google Scholar]
  29. Koldovský O., Asp N. G., Dahlqvist A. A method for the separate assay of "neutral" and "acid" beta-galactosidase in homogenates of rat small-intestinal mucosa. Anal Biochem. 1969 Mar;27(3):409–418. doi: 10.1016/0003-2697(69)90054-2. [DOI] [PubMed] [Google Scholar]
  30. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kozak M. Regulation of translation in eukaryotic systems. Annu Rev Cell Biol. 1992;8:197–225. doi: 10.1146/annurev.cb.08.110192.001213. [DOI] [PubMed] [Google Scholar]
  32. Lathe R., Vilotte J. L., Clark A. J. Plasmid and bacteriophage vectors for excision of intact inserts. Gene. 1987;57(2-3):193–201. doi: 10.1016/0378-1119(87)90122-3. [DOI] [PubMed] [Google Scholar]
  33. Leah R., Kigel J., Svendsen I., Mundy J. Biochemical and molecular characterization of a barley seed beta-glucosidase. J Biol Chem. 1995 Jun 30;270(26):15789–15797. doi: 10.1074/jbc.270.26.15789. [DOI] [PubMed] [Google Scholar]
  34. Leese H. J., Semenza G. On the identity between the small intestinal enzymes phlorizin hydrolase and glycosylceramidase. J Biol Chem. 1973 Dec 10;248(23):8170–8173. [PubMed] [Google Scholar]
  35. Lenman M., Falk A., Xue J., Rask L. Characterization of a Brassica napus myrosinase pseudogene: myrosinases are members of the BGA family of beta-glycosidases. Plant Mol Biol. 1993 Feb;21(3):463–474. doi: 10.1007/BF00028804. [DOI] [PubMed] [Google Scholar]
  36. Lojda Z., Slabý J., Kraml J., Kolínská J. Synthetic substrates in the histochemical demonstration of intestinal disaccharidases. Histochemie. 1973 Mar 26;34(4):361–369. doi: 10.1007/BF00306308. [DOI] [PubMed] [Google Scholar]
  37. Lorenz-Meyer H., Blum A. L., Haemmerli H. P., Semenza G. A second enzyme defect in acquired lactase deficiency: lack of small-intestinal phlorizin-hydrolase. Eur J Clin Invest. 1972 Aug;2(5):326–331. doi: 10.1111/j.1365-2362.1972.tb00658.x. [DOI] [PubMed] [Google Scholar]
  38. Lottaz D., Oberholzer T., Bähler P., Semenza G., Sterchi E. E. Maturation of human lactase-phlorizin hydrolase. Proteolytic cleavage of precursor occurs after passage through the Golgi complex. FEBS Lett. 1992 Nov 30;313(3):270–276. doi: 10.1016/0014-5793(92)81207-3. [DOI] [PubMed] [Google Scholar]
  39. Maiuri L., Raia V., Potter J., Swallow D., Ho M. W., Fiocca R., Finzi G., Cornaggia M., Capella C., Quaroni A. Mosaic pattern of lactase expression by villous enterocytes in human adult-type hypolactasia. Gastroenterology. 1991 Feb;100(2):359–369. doi: 10.1016/0016-5085(91)90203-w. [DOI] [PubMed] [Google Scholar]
  40. Maiuri L., Rossi M., Raia V., D'Auria S., Swallow D., Quaroni A., Auricchio S. Patchy expression of lactase protein in adult rabbit and rat intestine. Gastroenterology. 1992 Dec;103(6):1739–1746. doi: 10.1016/0016-5085(92)91429-8. [DOI] [PubMed] [Google Scholar]
  41. Maiuri L., Rossi M., Raia V., Garipoli V., Hughes L. A., Swallow D., Norén O., Sjöström H., Auricchio S. Mosaic regulation of lactase in human adult-type hypolactasia. Gastroenterology. 1994 Jul;107(1):54–60. doi: 10.1016/0016-5085(94)90060-4. [DOI] [PubMed] [Google Scholar]
  42. Mantei N., Villa M., Enzler T., Wacker H., Boll W., James P., Hunziker W., Semenza G. Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme. EMBO J. 1988 Sep;7(9):2705–2713. doi: 10.1002/j.1460-2075.1988.tb03124.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Naim H. Y., Jacob R., Naim H., Sambrook J. F., Gething M. J. The pro region of human intestinal lactase-phlorizin hydrolase. J Biol Chem. 1994 Oct 28;269(43):26933–26943. [PubMed] [Google Scholar]
  44. Naim H. Y., Lacey S. W., Sambrook J. F., Gething M. J. Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved, enzymatically active, and transport-competent protein. J Biol Chem. 1991 Jul 5;266(19):12313–12320. [PubMed] [Google Scholar]
  45. Naim H. Y., Lentze M. J. Impact of O-glycosylation on the function of human intestinal lactase-phlorizin hydrolase. Characterization of glycoforms varying in enzyme activity and localization of O-glycoside addition. J Biol Chem. 1992 Dec 15;267(35):25494–25504. [PubMed] [Google Scholar]
  46. Naim H. Y., Naim H. Dimerization of lactase-phlorizin hydrolase occurs in the endoplasmic reticulum, involves the putative membrane spanning domain and is required for an efficient transport of the enzyme to the cell surface. Eur J Cell Biol. 1996 Jul;70(3):198–208. [PubMed] [Google Scholar]
  47. Naim H. Y. Processing of human pro-lactase-phlorizin hydrolase at reduced temperatures: cleavage is preceded by complex glycosylation. Biochem J. 1992 Jul 1;285(Pt 1):13–16. doi: 10.1042/bj2850013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Naim H. Y., Sterchi E. E., Lentze M. J. Biosynthesis and maturation of lactase-phlorizin hydrolase in the human small intestinal epithelial cells. Biochem J. 1987 Jan 15;241(2):427–434. doi: 10.1042/bj2410427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Naim H. Y. The pro-region of human intestinal lactase-phlorizin hydrolase is enzymatically inactive towards lactose. Biol Chem Hoppe Seyler. 1995 Apr;376(4):255–258. [PubMed] [Google Scholar]
  50. Neele A. M., Einerhand A. W., Dekker J., Büller H. A., Freund J. N., Verhave M., Grand R. J., Montgomery R. K. Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis. Gastroenterology. 1995 Oct;109(4):1234–1240. doi: 10.1016/0016-5085(95)90583-9. [DOI] [PubMed] [Google Scholar]
  51. Nsi-Emvo E., Launay J. F., Raul F. Improved purification of rat intestinal lactase. Gen Physiol Biophys. 1986 Feb;5(1):53–59. [PubMed] [Google Scholar]
  52. Oberholzer T., Mantei N., Semenza G. The pro sequence of lactase-phlorizin hydrolase is required for the enzyme to reach the plasma membrane. An intramolecular chaperone? FEBS Lett. 1993 Oct 25;333(1-2):127–131. doi: 10.1016/0014-5793(93)80389-c. [DOI] [PubMed] [Google Scholar]
  53. Ouwendijk J., Moolenaar C. E., Peters W. J., Hollenberg C. P., Ginsel L. A., Fransen J. A., Naim H. Y. Congenital sucrase-isomaltase deficiency. Identification of a glutamine to proline substitution that leads to a transport block of sucrase-isomaltase in a pre-Golgi compartment. J Clin Invest. 1996 Feb 1;97(3):633–641. doi: 10.1172/JCI118459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Quaroni A., Isselbacher K. J. Study of intestinal cell differentiation with monoclonal antibodies to intestinal cell surface components. Dev Biol. 1985 Oct;111(2):267–279. doi: 10.1016/0012-1606(85)90482-8. [DOI] [PubMed] [Google Scholar]
  55. Rossi M., Maiuri L., Salvati V. M., Russomanno C., Auricchio S. Analysis of lactase processing in rabbit. FEBS Lett. 1993 Dec 27;336(2):299–303. doi: 10.1016/0014-5793(93)80824-e. [DOI] [PubMed] [Google Scholar]
  56. Skovbjerg H., Danielsen E. M., Noren O., Sjöström H. Evidence for biosynthesis of lactase-phlorizin hydrolase as a single-chain high-molecular weight precursor. Biochim Biophys Acta. 1984 Apr 10;798(2):247–251. doi: 10.1016/0304-4165(84)90312-x. [DOI] [PubMed] [Google Scholar]
  57. Trimbur D. E., Warren R. A., Withers S. G. Region-directed mutagenesis of residues surrounding the active site nucleophile in beta-glucosidase from Agrobacterium faecalis. J Biol Chem. 1992 May 25;267(15):10248–10251. [PubMed] [Google Scholar]
  58. Tull D., Withers S. G., Gilkes N. R., Kilburn D. G., Warren R. A., Aebersold R. Glutamic acid 274 is the nucleophile in the active site of a "retaining" exoglucanase from Cellulomonas fimi. J Biol Chem. 1991 Aug 25;266(24):15621–15625. [PubMed] [Google Scholar]
  59. Wacker H., Keller P., Falchetto R., Legler G., Semenza G. Location of the two catalytic sites in intestinal lactase-phlorizin hydrolase. Comparison with sucrase-isomaltase and with other glycosidases, the membrane anchor of lactase-phlorizin hydrolase. J Biol Chem. 1992 Sep 15;267(26):18744–18752. [PubMed] [Google Scholar]

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