LYSOSOMAL MEMBRANE TRANSPORT PHYSIOLOGICAL AND PATHOLOGICAL EVENTS Front cover: The choir gallery by Donatello, at the Museum of the Opera del Duomo (Firenze), can be imagined as a busy and lively biological membrane. LYSOSOMAL MEMBRANE TRANSPORT PHYSIOLOGICAL AND PATHOLOGICAL EVENTS LYSOSOMAAL MEMBRAAN TRANSPORT FYSIOLOGISCHE EN PATHOLOGISCHE PROCESSEN PROEFSCHRIFT TER VERKRUGING VAN DE GRAAD VAN DOCTOR AAN DE ERASMUS UNIVERSITEIT ROTTERDAM OP GEZAG VAN DE RECTOR MAGN!FICUS PROF. DR. C.J. RUNVOS EN VOLGENS BESLUIT VAN HET COLLEGE VAN DEKANEN. DE OPENBARE VERDEDIGING ZAL PLAATSVINDEN OP WOENSDAG 18 DECEMBER 1991 OM 13.45 UUR DOOR GRAZ!A MARIA SIMONETTA MANCINI geboren te San Severo PROMOTIECOMMISSIE Promotor: Prof. Dr. H. Galjaard Co-promotor: Dr. F.W. Verheijen Overige leden: Prof. Dr. P.P. Aula Prof. Dr. H.R. Scholte Prof. Dr. H.K.A. Visser Dit proefschrift werd bewerkt binnen de vakgroep Celbiologie en Genetica van de Faculteit der Geneeskunde en Gezondheidswetenschappen van de Erasmus Universiteit Rotterdam. Het onderzoek werd financieel gesteund door de Stichting Klinische Genetica regia Rotterdam. LYSOSOMAL MEMBRANE TRANSPORT PHYSIOLOGICAL AND PATHOLOGICAL EVENTS THESIS TO OBTAIN THE DEGREE OF DOCTOR AT THE ERASMUS UNIVERSITY OF ROTTERDAM BY AUTHORIZATION OF THE RECTOR MAGNIFICUS PROF. DR. C.J. RIJNVOS AND BY THE DECISION OF THE COLLEGE OF DEANS. THE PUBLIC DEFENSE WILL TAKE PLACE ON WEDNESDAY, DECEMBER 18"' 1991 AT 13.45 p.m. BY GRAZIA MARIA SIMONETTA MANCINI born in San Severo "Felix qui potuit rerum cognoscere causas. " (P. Vergilius Maro. Georgica) To all the children who cannot be cured by a few difficult words. CONTENTS: Introduction 11 Chapter 1: THE LYSOSOMAL MEMBRANE - Chemical structure: lipid composition 15 - Membrane proteins 16 - Integral lysosomal membrane proteins of unknown function 18 - lntegrallysosomal membrane enzymes 20 Chapter 2: LYSOSOMAL MEMBRANE TRANSPORTERS - Transport mechanisms 25 - Channels and pumps in the lysosome 29 - Lysosomal membrane carriers 33 - Amino acids 34 -Sugars 36 - Other substrates 37 - Discussion 40 Chapter 3: GENETIC DISORDERS OF LYSOSOMAL TRANSPORT 49 Chapter 4: SIALIC ACID STORAGE DISORDERS - Sialic acid metabolism 53 - Clinical aspects of sialic acid storage diseases 55 Chapter 5: THE EXPERIMENTAL WORK - lntroduction 63 - Publication I: Free sialic acid storage disease. A new Italian case. (Fois, A., Balestri, P., Fametani, M.A., Mancini, G.M.S., Borgogni, P., Margollicci, M.A., Molinelli, M., Alessandrini, C. and Gerli, R.; Eur. J. Pediat. 146:195-198, 1987) 71 - Publication II: Free N-acetylneuraminic acid (NAN A) storage disorders: evidence for defective NANA transport across the lysosomal membrane. (Mancini, G.M.S., Verheifen, F. W. and Galjaard, H.; Hum. Genet. 73:214-217, 1986) 77 - Publication lll: Characterization of a proton-driven carrier for sialic acid in the lysosomal membrane. Evidence for a group­ specific transport system for acidic monosaccharides. (Mancini, G.M.S., de Jonge, H.R., Galjaard, H. and Verheijen, F. W.; J. Bioi. Chern. 264:15247-15254, 1989) 83 - Publication N: Glucose transport in lysosomal membrane vesicles. Kinetic demonstration of a carrier for neutral hexoses. (Mancini, G. M.S., Beerens, C.E.M. T. and Verheijen, F. W.; J. Bioi. Chern. 265:12380-12387, 1990) 93 - Publication V: Sialic acid storage diseases. A multiple lysosomal transport defect for acidic monosaccharides. (Mancini, G.M.S., Beerens, C.E.M.T., Aula, P.P. and Verheijen, F. W.; J. Clin. Invest. 87:1329-1335, 1991) 103 - Publication VI: Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes. (Mancini, G.M.S., Beerens, C.E.M. T., Galjaard, H. and Verheijen, F. W.; Submitted) 113 - General discussion and future perspectives 127 Surnrnary 131 Samenvatting 133 Acknowledgments 135 Curriculum vitae 137 Introduction Lysosomes are intracellular acid organelles containing a wide variety of hydrolases and surrounded by a single membrane. Compared with the amount of knowledge available about the function and the genetic defects of the lysosomal hydrolases, the understanding of the function of the lysosome membrane in physiological and pathological processes is only at the beginning. All the information available has been gained in the last decade. Before that, the lysosome membrane has been considered to be only a mechanical border limiting the access of the acidic h ydrolases to the surrounding cytoplasmic substrates. This finds its explanation in the assumption that lysosomes represent essentially the "terminal degradative compartment" of the cell (Kornfeld and Mellman, 1989), and that their function is strictly linked to cellular catabolism. Recent work has shown that efflux and bioavailability of lysosomal degradation products is controlled by specific transport proteins. In addition, in this organelle, active metabolites (vitamines, antigens, hormones like tyroxine) are produced and released via specific carriers, before reaching their cytoplasmic target (Gahl, 1989; Harding eta!., 1991; Tietze et a!., 1989). Lysosomes can also represent a storage compartment for neurotransmitters (biogenic amines, in lysosome-like specialized organelles) or for substances (like Ca++ ions) (Lemons and Thoene, 1991; Nemere and Norman,!986), which are released under humoral control (Nemere and Norman, 1989; Nelson, 1991). Moreover, not only the efflux, but also the influx of low molecular weight metabolites into the lysosomes can represent a mechanism of regulation of metabolic functions. For example, lysosomal uptake 11 of cytosolic amino acids is thought to regulate autophagic proteolysis during starvation (Mortimore et al., 1988; Christensen, 1990). All these processes are under control of specific transporters in the lysosome membrane. Information about lysosomal membrane transport proteins, as described in this thesis, has contributed to new concepts about the availability of metabolites, which in tum are essential for the metabolism in the intracellular compartments. On the one hand, the study of cell material derived from patients with a single gene defect has enabled the identification of new transport mechanisms (cystinosis, sialic acid storage diseases, etc.). On the other hand, biochemical studies have revealed an unexpectedly large number of different types of transport systems which in some instances have formed the basis for the elucidation of the molecular defect(s) in hitherto unexplained genetic disorders. This thesis deals with the identification of a lysosomal transport mechanism for sialic acid. A primary genetic defect of this transport mechanism is demonstrated in patients affected by lysosomal storage of free sialic acid and excessive sialuria (sialic acid storage diseases, SASD). A new patient is described, who presents with a clinical form of the disease slightly different from that of the other two well defined phenotypes (Salla disease and the infantile sialic acid storage disease, respectively). The lysosomal sialic acid carrier works as a unique H+ -cotransport system. Unexpectedly, this carrier shows a wide substrate specificity for acidic monosaccharides, including, for example, glucuronic acid. The demonstration of a transport defect for both sialic acid and glucuronic acid in lysosomes of patients with SASD introduced the concept of multiple transport defect in lysosomal disorders. In addition, a distinct carrier for neutral hexoses has been characterized, and its normal function in patients with SASD has been demonstrated. The first successful functional reconstitution of the lysosomal acidic sugar transporter, achieved in the course of our research, represents an important step to enable its purification and detailed molecular characterization. REFERENCES * Christ~omsen, H.N. (1990) Role of amino acid transport and countertransport in nutrition and metabolism. Physiol. Rev. 70:43~77 * Gahl. W.A. (1989) Lysosomal membrane transport in cellular nutrition. Annu. Rev. Nurr. 9:39-61 * Harding, C. V., Collins, D.S., Slot, J. W .. Geuze, H.J. and Unanue, E.R. (1991) Liposome-encapsuled antigens are processed in lysosomes, recycled and presented toT cells. Cell 64:393-401 * Kornfeld, S. and I. Mellman (1989) The biogenesis of lysosomes. Annu. Rev. Cell Biol. 5:483-525 * Lemons. R.M. and Thoene, J.G. (1991) Mediated calcium transport by isolated human fibroblast lysosomes. J. Biol. Chem. 266:14378-14382 * Mortimore. G.E., Wert, J.J. and Adams. C.E. (1988) Regulationofmicroautophagy and basal protein turnover in rat liver. Effects of short term starvation. J. Biol. Chern. 263:19545-19551 * Nelson. N. (1991) Structure and pharmacology of the proton-ATPases. Trends in Physiol. Sci. 12:71- 75 12 * Nemere,I. and Norman, A.W. {1986) 1,25 DihydroxyvitaminD3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing Ca ..... and ea++ -binding protein (calbindin-D28K) J. Bioi. Chem. 261:16106-16114 * Nemere, I. and Norman, A. W. (1989) 1,25-DihydroxyvitaminD3-mediated vesicular calcium transport in intestine: dose-response studies. Mol. Cell. Endocri!UJL 67:47-53. * Tietze, F., Kahn, L.D., Kobn,A.D., Bernardini, I .. Andersson, H.C., Adamson. M.D.,Harper, G.S. and Gahl, W.A. (1989) Carrier-mediated transport of monoiodotyrosineout of thyroid celllysosomes. J. Bioi. Chem. 264:4762-4765 13 Chapter 1 THE LYSOSOMAL MEMBRANE Chemical structure: lipid composition The lysosomal membrane is constituted by a phospholipid bilayer, containing a large number of membrane-associated and integral-membrane proteins. The protein/lipid