University of Veterinary Medicine Hannover Department of Physiological Chemistry The concerted action of multiple post-translational events regulates the trafficking and function of wild type and mutant disaccharidases INAUGURAL DOCTORAL THESIS in partial fulfillment of the requirements of the degree of Doctor of Natural Sciences -Doctor rerum naturalium- (Dr. rer. nat.) submitted by Lena Diekmann, M.Sc. Bünde, Germany Hannover 2016 Supervisor: Prof. Dr. phil. nat. Hassan Y. Naim Department of Physiological Chemistry Institute for Biochemistry University of Veterinary Medicine Hannover, Germany Supervision group: Prof. Dr. phil. nat. Hassan Y. Naim Department of Physiological Chemistry Institute for Biochemistry University of Veterinary Medicine Hannover, Germany Prof. Dr. rer. nat. Georg Herrler Department of Infectious Diseases Institute of Virology University of Veterinary Medicine Hannover, Germany 1st Evaluation: Prof. Dr. phil. nat. Hassan Y. Naim Department of Physiological Chemistry Institute for Biochemistry University of Veterinary Medicine Hannover, Germany 2nd Evaluation: Prof. Dr. rer. nat. Rita Gerardy-Schahn Institute for Cellular Chemistry Hannover Medical School, Germany Date of the final exam: 19.04.2016 Dedicated to my family Table of contents I Table of contents Table of contents ....................................................................................................... I List of publications .................................................................................................. III Abbreviations ........................................................................................................... IV List of tables ........................................................................................................... VII List of figures .......................................................................................................... VII Abstract .................................................................................................................. VIII Zusammenfassung ................................................................................................... X Introduction ............................................................................................................... 1 Types of lactase deficiencies .................................................................................. 2 Symptoms of lactose intolerance and secondary associated disorders .......................... 7 Diagnosis of lactose malabsorption and lactose intolerance ........................................... 8 Treatment of lactose intolerance in infants, children and adults .....................................10 Lactase-phlorizin hydrolase (LPH) ........................................................................ 10 Biosynthesis and intracellular processing ......................................................................12 Polarized sorting ...........................................................................................................14 Protein modifications, folding and quality control in the ER ................................... 15 Aim of the dissertation ........................................................................................... 22 Publications ............................................................................................................ 23 Congenital lactose intolerance is triggered by severe mutations on both alleles of the lactase gene .................................................................................................... 25 The Diverse Forms of Lactose Intolerance and the Putative Linkage to Several Cancers ................................................................................................................. 27 Structural Determinants for transport of a multi-domain membrane glycoprotein in the early secretory pathway .................................................................................. 28 II Table of contents Discussion .............................................................................................................. 66 Difficulties in the diagnosis and possible molecular causes of the low lactase activity in CLD patients .......................................................................................... 66 Identification and molecular analysis of two novel mutations of the LCT gene causing CLD .......................................................................................................... 68 Characterization and implication of the subdomains of LPH, a multi-domain protein, on its function and folding ...................................................................................... 74 Conclusion .............................................................................................................. 79 References .............................................................................................................. 80 Acknowledgements .............................................................................................. 106 Eidesstattliche Erklärung ..................................................................................... 107 List of publications III List of publications Parts of this thesis were already published/are under revision Diekmann L., Pfeiffer K., and Naim H. Y., Congenital lactose intolerance is triggered by severe mutations on both alleles of the lactase gene. BMC Gastroenterol. 2015 Mar 21;15:36. DOI: 10.1186/s12876-015-0261-y. Amiri M.*, Diekmann L.*, von Köckritz-Blickwede M. and Naim H. Y., The Diverse Forms of Lactose Intolerance and the Putative Linkage to Several Cancers. Nutrients. 2015 Aug 28;7(9):7209-30. DOI: 10.3390/nu7095332. Diekmann L.*, Behrendt M.*, Amiri M. and Naim H. Y., Structural determinants for transport of a multi-domain membrane glycoprotein in the early secretory pathway, J Biol Chem., under revision Publication (not relevant for this thesis) Maria Henström*, Lena Diekmann*, … , Hassan Y. Naim° Mauro and D’Amato°, Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome, NEJM, in progress *° Authors contributed equally Conference contributions regarding this thesis Lena Diekmann, Katrin Pfeiffer, and Hassan Naim, Compound heterozygous mutations elicit congenital lactase deficiency in a Japanese infant, FASEB J April 2015 29:596.5 IV Abbreviations Abbreviations ATH adult type of hypolactasia ATP adenosine triphosphate BiP binding immunoglobulin protein bp base pairs BSA bovine serum albumin °C degree Celsius cDNA complementary DNA CLD congenital lactase deficiency CNX calnexin COS-1 african green monkey kidney fibroblast-like cells CRT calreticulin CSID ongenital sucrase-isomaltase deficiency DEAE diethyle-amino-ethyle del deletion DMEM Dulbecco´s modified Eagle Medium DNA desoxyribonucleid acid DTT dithiothreitol e.g. (exempli gratia) for example EDEM ER degradation-enhancing α-mannosidase-like protein endo H endo-β-N-acetylglucosaminidase H ER endoplasmic reticulum ERAD ER-associated degradation et al. et alii (and others) FCS fetal calf serum fs frameshift x g acceleration of gravity GABA γ-aminobutyric acid GH glycoside hydrolase GlcNAc N-acetylglucosamine GPI glycosylphosphatidylinositol Abbreviations V h hour/hours Hsp70 heat shock protein 70 IBS irritable bowel syndrome IP immunoprecipitation kbp kilobase pair kDa kilo Dalton KLD Kongenitale Laktase Defizienz LCT lactase gene LPH lactase-phlorizin hydrolase m milli (10-3) M molar mass µ micro (10-6) mAbs monoclonal antibodies MEM methionine-free minimum essential medium MGAM maltase-glucoamylase min minute/minutes mRNA messenger RNA NMD nonsense-mediated mRNA decay PDI protein disulfide isomerase PNGase F peptide-N-Glycosidase F RNA ribonucleic acid RT room temperature SDS sodium dodecyl sulfate SERCA sarco(endo)plasmic reticulum Ca2+ ATPase SI sucrase-isomaltase SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis sec seconds SGLT1 sodium/glucose co-transporter 1 SNPs single nucleotide polymorphisms TEMED tetramethylethylenediamine TGN trans-Golgi network VI Abbreviations TRIS Tris(hydroxymethyl)aminomethan UGGT UDP-glucose:glycoprotein glucosyltransferase UPF1 up-frameshift protein 1 UPR unfolded protein response w/v weight/volume X stop codon Amino acid Three letter code One letter code alanine ala A arginine arg R asparagine asn N aspartic acid asp D asparagine or aspartic acid asx B cysteine cys C glutamic acid glu E glutamine gln Q glutamine or glutamic acid glx Z glycine gly G histidine his H isoleucine ile I leucine leu L lysine lys K methionine met M phenylalanine phe F proline pro P serine ser S threonine thr T tryptophan trp W tyrosine tyr Y valine val V List of tables VII List of tables Table 1: Different types of lactase deficiencies ........................................................... 3 Table 2: Reported CLD patients with mutations in the LCT gene ............................... 6 List of figures Figure 1: Maturation steps of LPH in the intestinal epithelial cells. ........................... 14 Figure 2: Structure of the N-linked core glycan. ........................................................ 17 Figure 3: Quality control of newly synthesized proteins in the endoplasmic reticulum. ................................................................................................................................. 19 Figure 4: Potential requirements for heterodimerization of LPH wild type with a pathogenic mutant.