Thomas Kolter

Sphingolipids—Their Metabolic Pathways and the Pathobiochemistry of Neurodegenerative Diseases

Glycolipids such as ganglioside GM1 are involved in the building of carbohydrate layers on the surface of living cells. The investigation of the metabolism of this class of compounds gives insight into human diseases, novel signal transduction processes, and the epidermal water permeability barrier.

Lysosomal degradation of membrane lipids

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid‐binding proteins, the Niemann–Pick disease, type C2‐protein, the GM2‐activator, and the saposins sap‐A, ‐B, ‐C, and ‐D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes.

Topology of glycosphingolipid degradation.

Glycosphingolipids (GSLs) form cell-type-specific patterns on the surface of eukaryotic cells. Degradation of plasma-membrane-derived GSLs in the lysosomes after internalization through the endocytic pathway is achieved through the concerted actions of hydrolysing enzymes and sphingolipid activator proteins. The latter are proteins necessary for the degradation of GSLs possessing short oligosaccharide chains. Some activator proteins bind to GSLs and form water-soluble complexes, which lift out of the membrane and give the water-soluble hydrolysing enzymes access to the regions of the GSL that would otherwise be obscured by the membrane. The inherited deficiency of both lysosomal hydrolases and sphingolipid activator proteins gives rise to sphingolipid storage diseases. An analysis of these diseases suggests a new model for the topology of endocytosis and lysosomal digestion, which is discussed in this article.

Principles of lysosomal membrane degradation Cellular topology and biochemistry of lysosomal lipid degradation

Cellular membranes enter the lysosomal compartment by endocytosis, phagocytosis, or autophagy. Within the lysosomal compartment, membrane components of complex structure are degraded into their building blocks. These are able to leave the lysosome and can then be utilized for the resynthesis of complex molecules or can be further degraded. Constitutive degradation of membranes occurs on the surface of intra-endosomal and intra-lysosomal membrane structures. Many integral membrane proteins are sorted to the inner membranes of endosomes and lysosome after ubiquitinylation. In the lysosome, proteins are degraded by proteolytic enzymes, the cathepsins. Phospholipids originating from lipoproteins or cellular membranes are degraded by phospholipases. Water-soluble glycosidases sequentially cleave off the terminal carbohydrate residues of glycoproteins, glycosaminoglycans, and glycosphingolipids. For glycosphingolipids with short oligosaccharide chains, the additional presence of membrane-active lysosomal lipid-binding proteins is required. The presence of lipid-binding proteins overcomes the phase problem of water soluble enzymes and lipid substrates by transferring the substrate to the degrading enzyme or by solubilizing the internal membranes. The lipid composition of intra-lysosomal vesicles differs from that of the plasma membrane. To allow at least glycosphingolipid degradation by hydrolases and activator proteins, the cholesterol content of these intraorganellar membranes decreases during endocytosis and the concentration of bis(monoacylglycero)phosphate, a stimulator of sphingolipid degradation, increases. A considerable part of our current knowledge about mechanism and biochemistry of lysosomal lipid degradation is derived from a class of human diseases, the sphingolipidoses, which are caused by inherited defects within sphingolipid and glycosphingolipid catabolism.

Growth promoting and metabolic activity of the human insulin analogue [GlyA21,ArgB31,ArgB32]insulin (HOE 901) in muscle cells

[GlyA21,ArgB31,ArgB32]insulin (HOE 901) represents a biosynthetic human insulin analogue that, due to its isoelectric point, precipitates at neutral tissue pH leading to a retarded absorption rate and a corresponding longer duration of action. In the present investigation we have evaluated the growth promoting and metabolic activity of this analogue in muscle tissue using exponentially growing H9c2 cardiac myoblasts and adult rat ventricular cardiomyocytes. Equilibrium binding studies of 125I-labelled IGF-I (insulin-like growth factor I) to differentiating myoblasts revealed the presence of 7 x 10(3) IGF-I receptors per cell. In contrast, no specific binding of insulin could be detected. Competition binding experiments showed a slightly higher affinity of HOE 901 for the IGF-I receptor when compared to regular human insulin with IC50 (half-inhibitory concentration) values of 70 and 101 nM, respectively. However, the supermitogenic insulin analogue [AspB10]insulin competed significantly more efficiently for IGF-I binding (IC50: 44 nM). Maximum growth promoting activity of the peptides was then determined in serum-starved myoblasts by an incubation with the peptides (5 x 10(-7) M) for 16 h in the presence of [3H]thymidine. [Asp(B10)]Insulin produced a stimulation of DNA synthesis (about 3-fold) which was comparable to the effect of IGF-I and significantly (P < 0.005) higher than the effect of HOE 901 with the latter being essentially equipotent to native insulin. Comparable results were obtained at lower concentrations of the peptides (10(-9) to 10(-8) M). Metabolic activity of HOE 901 was determined by measuring the dose-dependent stimulation of 3-O-methylglucose transport in adult cardiomyocytes. Maximum transport stimulation was identical for insulin and HOE 901 with EC50 (half-effective concentration) values of 0.7 x 10(-10) and 1.9 x 10(-9) M, respectively. We concluded that the IGF-I receptor-mediated growth promoting activity of HOE 901 in muscle cells and the maximal metabolic activity of this analogue are not different from those of native human insulin. It is suggested that differential interaction with IGF-I receptors significantly contributes to the action profile of insulin analogues.

Recent advances in the biochemistry of sphingolipidoses.

Glycosphingolipids are ubiquitous membrane components of eukaryotic cells. They participate in various cell recognition events and can regulate enzymes and receptors within the plasma membrane. Sphingolipidoses are due to an impaired lysosomal digestion of these substances. Glycosphingolipids are degraded by the action of exohydrolases, which are supported, in the case of glycosphingolipids with short oligosaccharide chains, by sphingolipid activator proteins. Five sphingolipid activator proteins are known so far, the GM2‐activator and the SAPs, SAP‐A to D (also called saposins). Degradation of glycosphingolipids requires endocytic membrane flow of plasma membrane derived glycosphingolipids into the lysosomes. Recent research focused on the topology of this process and on the mechanism and physiological function of sphingolipid activator proteins. Limited knowledge is available about enzymology and topology of glycosphingolipid biosynthesis. Recently, intermediates of this metabolic pathway have been identified as novel signalling molecules. Inhibition of glycosphingolipid biosynthesis has been shown to be beneficial in the animal model of Tay‐Sachs disease. Mice with disrupted genes for lysosomal hydrolases and activator proteins are useful models for known human diseases and are valuable tools for the study of glycosphingolipid metabolism, the pathogenesis of sphingolipidoses and novel therapeutic approaches.

A view on sphingolipids and disease

Sphingolipid and glycosphingolipid levels and expression of sphingolipid metabolizing enzymes are altered in a variety of diseases or in response to drug treatment. Inherited defects of enzymes and other proteins required for the lysosomal degradation of these lipids lead to human sphingolipidoses. Also genetic defects that affect sphingolipid biosynthesis are known. Although the molecular details are often far from clear, (glyco)sphingolipids have been implicated to play a role in atherosclerosis, insulin resistance, cancer, and infections by pathogens. More general aspects of selected diseases are discussed.

Chemical chaperones--a new concept in drug research.

Low-molecular-weight compounds are able to stabilize the conformation of proteins that are defective in patients of inherited diseases. Unspecifically acting chemical chaperones, including osmolytes, can increase the fraction of the correctly folded variant protein encoded by the mutated gene. More recently, the concept of specifically acting chemical chaperones has been applied to two sphingolipid storage diseases, Fabrys disease (Figure 1) and Gauchers disease (Figure 2). Studies in cultured cells and, in the case of Fabrys disease also in the animal model, revealed that administration of inhibitors led to a significant increase in the activity of the variant enzymes and to a substantial improvement of therapeutic parameters.

Normal phase liquid chromatography coupled to quadrupole time of flight atmospheric pressure chemical ionization mass spectrometry for separation, detection and mass spectrometric profiling of neutral sphingolipids and cholesterol.

Many lipidomic approaches focus on investigating aspects of sphingolipid metabolism. Special emphasis is put on neutral sphingolipids and cholesterol and their interaction. Such an interest is attributed to the fact that those lipids are altered in a series of serious disorders including various sphingolipidoses. High performance thin-layer chromatography (HPTLC) has become a widely used technique for lipid analysis. However, mass spectrometric profiling is irreplaceable for gaining an overview about the various molecular species within a lipid class. In this work we have developed a sensitive method based on a gradient normal phase high performance liquid chromatography (HPLC) coupled to quadrupole time of flight (QTOF) atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in positive mode, which for the first time enables separation, on-line detection, and mass spectrometric profiling of multiple neutral sphingolipids including ceramide, glucosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin as well as cholesterol within less than 15min. An important advantage of the presented HPLC/APCI-MS approach is that the separation pattern emulates the one obtained by an optimized HPTLC method with a multiple stage development. Thus, the lipid classes previously separated and quantified by HPTLC can be easily screened regarding their mass spectrometric profiles by HPLC/APCI-MS. In addition, the selected ionization conditions enable in-source fragmentation providing useful structural information. The methods (HPLC/APCI-MS and the optimized HPTLC) were applied for the analysis of the mentioned lipids in human fibroblasts. This approach is aimed basically at investigators who perform studies based on genetic modifications or treatment with pharmacological agents leading to changes in the biochemical pathways of neutral sphingolipids and cholesterol. In addition, it can be of interest for research on disorders related to impairments of sphingolipid metabolism.

Sphingolipide – ihre Stoffwechselwege und die Pathobiochemie neurodegenerativer Erkrankungen

Glycolipide wie das Gangliosid GM1 sind am Aufbau der Kohlenhydratschicht beteiligt, die die Oberflache lebender Zellen bedeckt. Untersuchungen des Stoffwechsels dieser Verbindungsklasse lieferten Erkenntnisse uber menschliche Erbkrankheiten, neuartige Signaltransduktionsprozesse und die Wasserpermeabilitatsbarriere der Haut.