Biomedical subjects
Thomas Kolter
Publications and source records attributed to Thomas Kolter.
Sphingolipid metabolism diseases.
Human diseases caused by alterations in the metabolism of sphingolipids or glycosphingolipids are mainly disorders of the degradation of these compounds. The sphingolipidoses are a group of monogenic inherited diseases caused by defects in the system of lysosomal sphingolipid degradation, with subsequent accumulation of non-degradable storage material in one or more organs. Most sphingolipidoses are associated with high mortality. Both, the ratio of substrate influx into the lysosomes and the reduced degradative capacity can be addressed by therapeutic approaches. In addition to symptomatic treatments, the current strategies for restoration of the reduced substrate degradation within the lysosome are enzyme replacement therapy (ERT), cell-mediated therapy (CMT) including bone marrow transplantation (BMT) and cell-mediated "cross correction", gene therapy, and enzyme-enhancement therapy with chemical chaperones. The reduction of substrate influx into the lysosomes can be achieved by substrate reduction therapy. Patients suffering from the attenuated form (type 1) of Gaucher disease and from Fabry disease have been successfully treated with ERT.
Dopaminergic modulation of semantic priming in healthy volunteers.
BACKGROUND: Semantic priming is a function related to prefrontal cortical (PFC) networks and is lateralized. There is evidence that semantic priming underlies dopaminergic modulation. It is known that the D1-receptor is more abundant in prefrontal networks; however, until now there have been no studies investigating the selective modulation of semantic priming with dopamine agonists. Furthermore, D1 receptor dysfunction has been described in schizophrenia, and patients with formal thought disorder seem to have disturbed focusing of associations and increased indirect priming. METHODS: With a subtraction design, we compared the influence of pergolide (D1/D2 agonist) with bromocriptine (D2 agonist) and placebo, in a randomized, double-blind, crossover design in 40 healthy male volunteers. Subjects performed a lateralized lexical decision task including direct and indirect related prime-target pairs (stimulus onset asynchrony = 750 msec). RESULTS: Only on pergolide a decrease of the indirect priming in the left hemisphere presentations was found. CONCLUSIONS: These findings point to a potential selective modulation of agonists with a D1 component on the focusing of semantic associations. The clinical relevance of this study is that it might help the development of therapeutic strategies for treating cognitive deficits in schizophrenia and Parkinson's disease, which are highly relevant to the functional outcome.
The enzyme-binding region of human GM2-activator protein.
The GM2-activator protein (GM2AP) is an essential cofactor for the lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (HexA). It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interface. Functional deficiencies in this protein result in a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. In order to elucidate this cofactor's mode of action and identify the surface region of GM2AP responsible for binding to HexA, we designed several variant forms of this protein and evaluated the consequences of these mutations for lipid- and enzyme-binding properties using a variety of biophysical and functional studies. The point mutants D113K, M117V and E123K showed a drastically decreased capacity to stimulate HexA-catalysed GM2 degradation. However, surface plasmon resonance (SPR) spectroscopy showed that the binding of these variants to immobilized lipid bilayers and their ability to solubilize lipids from anionic vesicles were the same as for the wild-type protein. In addition, a fluorescence resonance energy transfer (FRET)-based assay system showed that these variants had the same capacity as wild-type GM2AP for intervesicular lipid transfer from donor to acceptor liposomes. The concentration-dependent effect of these variants on hydrolysis of the synthetic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-beta-D-glucopyranoside (MUGS) indicated a weakened association with the enzyme's alpha subunit. This identifies the protein region affected by these mutations, the single short alpha helix of GM2AP, as the major determinant for the interaction with the enzyme. These results further confirm that the function of GM2AP is not restricted to a biological detergent that simply disrupts the membrane structure or lifts the substrate out of the lipid plane. In contrast, our data argue in favour of the critical importance of distinct activator-hexosaminidase interactions for GM2 degradation, and corroborate the view that the activator/lipid complex represents the true substrate for the degrading enzyme.
Lipid-binding proteins in membrane digestion, antigen presentation, and antimicrobial defense.
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Site-specific cleavage--a model system for the identification of lipid-modified glutamate residues in proteins.
Numerous proteins are modified post-translationally after their biosynthesis at the ribosomes of the cell. One such modification, only poorly characterized to date, is the formation of lipid esters of glutamate side chains in the skin proteins of land-living mammals; here a subset of very long chain fatty acids, ceramides and/or glucosylceramides, are bound through their omega-hydroxy groups to structural proteins of the so-called "cornified envelope" in the outermost layer of the skin, the stratum corneum. We report an approach for the identification of proteins containing ester-modified glutamic acid residues and the determination of their positions within the peptide sequence, designed for mass spectrometric investigation of human skin proteins.
Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.
Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family.
Differential dopaminergic modulation of executive control in healthy subjects.
RATIONALE: Executive control (EC) has different subcomponents, e.g., response inhibition (measured, for example, by the Stroop task) and working memory (WM-measured, for example, by delayed response tasks, DRT). EC has been associated with networks involving the prefrontal cortex (PFC). Moreover, there is evidence that dopamine agonists, especially those with a D1 profile, may modulate EC, since in the PFC D1 subtype receptors are more abundant. OBJECTIVE: This study aimed to selectively distinguish whether D1 versus D2 dopamine agonism differentially influences EC related to the inhibition of irrelevant information and WM. Because of its D1 component, we predicted that the administration of pergolide (mixed D1/D2 agonist), in comparison with bromocriptine (D2 selective agonist) and placebo, would enhance performance in both EC tasks. Using a lateralized Stroop task, we predicted a decrease in the interference effect, as well as error rates, while no increase in facilitation effects. For the DRT task, we predicted fewer error scores in the delay condition. METHODS: Forty male healthy subjects participated in this randomized, double-blind, placebo-controlled, crossover study. RESULTS: For the Stroop task no superiority of pergolide was found; however, with bromocriptine, decreased interference was found. No modulation of lateralization effects was shown in interference measures. Moreover, subjects on pergolide showed an absence of facilitation effects. No effects of either agonist were found for the DRT. CONCLUSION: Our findings suggest that dopamine agonists modulate two EC tasks differently. Furthermore, there seems to be a selective modulation of different aspects of the Stroop task.
Photoaffinity labelling of the human GM2-activator protein. Mechanistic insight into ganglioside GM2 degradation.
The GM2-activator protein (GM2AP) is an essential cofactor for the degradation of ganglioside GM2 by lysosomal beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-bound substrate at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. To elucidate the mode of action of this glycoprotein cofactor, we synthesized the two photoaffinity labels [14C]C3-TPD-GM2 and [14C]C7-TPD-GM2. Incubation of GM2AP with these substrate analogues and subsequent irradiation led to covalent labelling of the protein. After separation of tryptic peptides by reverse-phase HPLC, the labelled peptide fractions were analysed by MALDI-TOF and sequenced by ESI-Q-TOF mass spectrometry. Both labels were found to be specifically photoincorporated into a part of the surface loop comprising residues V153-L163, a stretch of amino acids that was previously identified as the most flexible region in the crystal structure of the activator. Our results provide strong evidence that this loop constitutes the part of the activator protein that directly interacts with the ganglioside substrate, suggesting that the hydrophobicity and the great structural mobility of this element are crucial for the extraction of the membrane-embedded glycolipid, its stabilization inside the spacious cavity and its guidance to the enzyme's active site. This study demonstrates that the approach of photoaffinity labelling in conjunction with accurate mass measurements can provide insight into substrate binding interactions that complements structural information.
Sticky proteins for stem cells.
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Crystal structures of O-acetylated 2-acylamino-2-deoxy-D-galactose derivatives.
The X-ray structures of 1,3,4,6-tetra-O-acetyl-2-deoxy-alpha-D-galactopyranoside derivatives with four different 2-(acylamino) substituents have been determined with Mo K(alpha) radiation at 123 K. The structure of the 2-acetylamino derivative and of its acyl-homologs with a 2-(propanoylamino)-, 2-(butanoylamino)-, and 2-(2-methyl-propanoylamino)-group crystallized in the monoclinic space group C2. The pyranose unit of all compounds has the usual 4C(1) shape. The different orientations of the 6-O-acetyl-groups are discussed. Conformations of the acylamino-group are compared to those found in the crystal structure of N-acetyl-alpha-D-galactosamine.
Inhibitors of endocannabinoid degradation: potential therapeutics for neurological disorders.
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Biosynthesis and degradation of mammalian glycosphingolipids.
Glycolipids are a large and heterogeneous family of sphingolipids that form complex patterns on eukaryotic cell surfaces. This molecular diversity is generated by only a few enzymes and is a paradigm of naturally occurring combinatorial synthesis. We report on the biosynthetic principles leading to this large molecular diversity and focus on sialic acid-containing glycolipids of the ganglio-series. These glycolipids are particularly concentrated in the plasma membrane of neuronal cells. Their de novo synthesis starts with the formation of the membrane anchor, ceramide, at the endoplasmic reticulum (ER) and is continued by glycosyltransferases of the Golgi complex. Recent findings from genetically engineered mice are discussed. The constitutive degradation of glycosphingolipids (GSLs) occurs in the acidic compartments, the endosomes and the lysosomes. Here, water-soluble glycosidases sequentially cleave off the terminal carbohydrate residues from glycolipids. For glycolipid substrates with short oligosaccharide chains, the additional presence of membrane-active sphingolipid activator proteins (SAPs) is required. A considerable part of our current knowledge about glycolipid degradation is derived from a class of human diseases, the sphingolipidoses, which are caused by inherited defects within this pathway. A new post-translational modification is the attachment of glycolipids to proteins of the human skin.
(4S,5S)-4-[(1R)-1,2-Dihydroxyethyl]-5-tridecyl-1,3-oxazolidin-2-one.
The title compound, C(18)H(35)NO(4), is a new bioactive amphiphilic lipid with a cis-substituted 1,3-oxazolidin-2-one head group. In the crystal structure, the molecules form intercalating bilayers in which the oxazolidinone head groups are joined together by hydrogen bonds into chains.
Chemical chaperones--a new concept in drug research.
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Recombinant ganglioside GM2 synthase--expression in insect cells and enzyme assay.
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Combinatorial ganglioside biosynthesis.
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Implicit and explicit learning in schizophrenics treated with olanzapine and with classic neuroleptics.
OBJECTIVE: Novel and classic neuroleptics differ in their effects on limbic striatal/nucleus accumbens (NA) and prefrontal cortex (PFC) dopamine turnover, suggesting differential effects on implicit and explicit learning as well as on anhedonia. The present study investigates whether such differences can be demonstrated in a naturalistic sample of schizophrenic patients. METHODS: Twenty-five inpatients diagnosed with DSM-IV schizophrenic psychosis and treated for at least 14 days with the novel neuroleptic olanzapine were compared with 25 schizophrenics taking classic neuroleptics and with 25 healthy controls, matched by age and education level. PFC/NA-dependent implicit learning was assessed by a serial reaction time task (SRTT) and compared with cerebellum-mediated classical eye-blink conditioning and explicit visuospatial memory. Anhedonia was measured with the Snaith-Hamilton-Pleasure Scale (SHAPS). RESULTS: Implicit (SRTT) and psychomotor speed, but not explicit (visuospatial) learning were superior in the olanzapine-treated group as compared to the patients on classic neuroleptics. Compared to healthy controls, olanzapine-treated schizophrenics showed similar implicit learning, but reduced explicit (visuospatial) memory performance. Acquisition of eyeblink conditioning was not different between the three groups. There was no difference with regard to anhedonia and SANS scores between the patients. CONCLUSION: Olanzapine seems to interfere less with unattended learning and motor speed than classical neuroleptics. In daily life, this may translate into better adaptation to a rapidly changing environment. The effects seem specific, as in explicit learning and eyeblink conditioning no difference to classic NL was found.