Guido Kohla

Functional anatomy of human lacrimal duct epithelium.

Abstract Resorption of tear fluid in the lacrimal ducts has hitherto been controversial; one reason for this has been insufficient knowledge of the anatomical structure and function of the lacrimal duct epithelium. The present study analyzes the structure of lacrimal duct epithelium by means of histological, histochemical, immunohistochemical and electronmicroscopical methods and draws a conclusion about its physiological function regarding its role in immunodeficiency. Investigations were performed on 31 lacrimal systems of 17 male and 14 female individuals (aged 54–88 years). Lacrimal ducts are surrounded by a wide-ranging cavernous system, which is embedded in an osseous canal between the maxilla and the lacrimal bone. The internal wall of the lacrimal canaliculi is lined by a stratified epithelium. The lacrimal sac and nasolacrimal duct contain a double-layered epithelium, which rests on a broad basement membrane. In their apical part epithelial cells contain large lipid droplets and secretory vacuoles. Epithelial cells are faced by microvilli and some tufts of kinociliae are also visible. Goblet cells are integrated in the epithelium as solitary cells or in a characteristical arrangement of several cells. The secretory product of these cells contains carbohydrates including fucose and sialic acid. Inside the surrounding cavernous system serous glands are found that open their excretory ducts into the lacrimal sac and nasolacrimal duct. Some T- and B-lymphocytes and macrophages may be demonstrated immunohistochemically in the submucosa partly penetrating the epithelium. Synthesized mucins of goblet cells form a specialized protective layer on the epithelium of the lacrimal ducts, which functionally serves for a simplified drainage of tear fluid into the inferior meatus of the nose. Together with immunocompetent cells, the protective layer plays a role in antigen defense and prevents invasion of pathogenic agents. The facing of epithelial cells by microvilli gives hints of reabsorption of lacrimal fluid inside the lacrimal ducts.

Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII

INTRODUCTION Host cell lines used for recombinant protein expression differ in their ability to perform post-translational modifications (PTMs). The currently available recombinant human FVIII (rhFVIII) products are produced in mammalian, non-human cell lines. For rhFVIII, glycosylation and sulfation are vital for functionality and von Willebrand factor (VWF)-binding affinity. Here we present the characterisation of the PTMs of a novel, human cell line-derived recombinant human FVIII (human-cl rhFVIII). rhFVIII expression in a human cell line avoids expression of undesirable mammalian glycoforms like Galα1-3Galβ1-GlcNAc-R (α-Gal) and N-glycolylneuraminic acid (Neu5Gc), which constitute epitopes antigenic to humans. MATERIALS AND METHODS We describe sulfation analysis, glycan profiling and characterisation using liquid chromatography-mass spectrometry and high performance anion exchange chromatography with pulsed amperometric detection. RESULTS AND CONCLUSIONS Human-cl rhFVIII is confirmed to be sulfated and glycosylated comparable to human plasma-derived FVIII. Most importantly, human-cl rhFVIII is devoid of the antigenic Neu5Gc or α-Gal epitopes observed in Chinese Hamster Ovary- and Baby Hamster Kidney-derived rFVIII products. Both the avoidance of non-human glycan structures and the achievement of complete sulfation are proposed to lower the intrinsic immunogenicity of human-cl rhFVIII compared with current rFVIII products.

Gangliosides with O-Acetylated Sialic Acids in Tumors of Neuroectodermal Origin

Gangliosides, carrying an O-acetylated sialic acid in their carbohydrate moiety, are often found in growing and developing tissues, especially of neuro-ectodermal origin. The most prominent one is 9-O-Ac-GD3, which is considered as an oncofetal marker in animal and human tumors like neuronal tumors, melanoma, basalioma or breast cancer, as well as in psoriatic lesions. Also other gangliosides like GD2 or GT3 were found to be O-acetylated in their terminal sialic acid. In this review we are summarising the occurrence of such gangliosides in normal and transformed tissues and delineate a more general theory that O-acetylated sialic acids in gangliosides are a universal marker for growing cells and tissues.

Metabolism and Role of O-Acetylated Sialic Acids

In the still growing family of sialic acids with more than 40 different derivatives of neuraminic acid, the interest of the scientific community is increasingly directed towards the O-acetylated species. In the past two decades it evolved that O-acetylated sialic acids play fundamental roles in the development of organisms, in the regulation of the immune system, in cancer processes and many other biological and pathophysiological events.

The human false vocal folds: an analysis of antimicrobial defense mechanisms

Inflammatory processes often lead to pathologic changes in the area of the larynx. A moistening function of the false vocal folds has been described frequently. Up to now we have little knowledge of the role of the false vocal folds in protection against pathogenic agents. The present study analyzes the structures of the false vocal folds in their relations to antimicrobial defense mechanisms. Investigations were performed on false vocal folds of larynges from 34 cadavers using histologic, histochemical and immunohistochemical methods. Seromucous glands, together with epithelial and goblet cells of the folds, synthesize a complex mucus layer. In all of the investigated samples this layer contains carbohydrates including N-acetyl-glucosamine, N-acetyl-galactosamine, galactose, mannose, fucose, and sialic acids. Furthermore, antimicrobial peptides like lactoferrin, lysozyme, alpha and beta defensins are also found in these structures. IgA, produced by plasma cells in the false vocal folds, is frequently integrated in the secretory product. Synthesized mucins, antimicrobial peptides and immunoglobulins form a specialized protective substance that is secreted mainly at the true vocal folds. Here the layer functions to lubricate the true vocal folds, resulting in positive functional consequences during vocal production. Moreover, together with immunocompetent cells, the protective layer seems to play a major role in antigen defense and prevents invasion of pathogenic agents.

Solubilisation and Properties of the Sialate-4-O-Acetyltransferase from Guinea Pig Liver

Abstract The O-acetylation of sialic acids turns out to be one of the most important modifications that influence the diverse biological and pathophysiological properties of glycoconjugates in animals and microorganisms. To understand the functions of this esterification, knowledge of the properties, structures and regulation of expression of the enzymes involved is essential. Attempts to solubilise, purify or clone the gene of one of the sialate-O-acetyltransferases have failed so far. Here we report on the solubilisation of the sialate 4-O-acetyltransferase from guinea pig liver, the first and essential step in the purification and molecular characterisation of this enzyme, by the zwitterionic detergent CHAPS. This enzyme Oacetylates sialic acids at C-4 both free and bound to oligosaccharides, glycoproteins and glycolipids with varying activity, however, gangliosides proved to be the best substrates. Correspondingly, a rapid enzyme test was elaborated using the ganglioside GD3. The soluble Oacetyltransferase maximally operated at 30C, pH 5.6, and 50-70 mM KCl and K2HPO4 concentrations. The Km values were 3.6 M for AcCoA and 1.2 M for GD3. CoA inhibits the enzyme with a Ki value of 14.8 M. A most important discovery enabling further enzyme purification is its need for an unknown low molecular mass and heat-stable cofactor that can be separated from the crude enzyme preparation by 30 kDa ultrafiltration.

Localisation and distribution of O-acetylated N-acetylneuraminic acids, the endogenous substrates of the hemagglutinin-esterases of murine coronaviruses, in mouse tissue

Infections by mouse hepatitis viruses result in disease of the liver, the gastrointestinal tract, respiratory tract, and the central nervous system. Coronaviruses related to mouse hepatitis virus express a hemagglutinin-esterase surface glycoprotein, which specifically hydrolyses either 5-N-acetyl-4-O-acetyl neuraminic acid (Neu4,5Ac2) or 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). Moreover, these sialic acids represent potential cellular receptor determinants for murine coronaviruses. Until now, the distribution of these sialic acids in mouse brain was not thoroughly investigated. Particularly Neu4,5Ac2 was not yet found in mouse brain. Using a sensitive method of gas chromatography coupled to mass spectrometry in the electron impact mode of ionization this manuscript demonstrates the occurrence of 13 different sialic acids varying in their alkyl and acyl substituents in mouse tissues including 5-N-acetyl-4-O-acetyl-9-O-lactyl-neuraminic acid (Neu4,5Ac29Lt), 5-N-acetyl-9-O-lactyl-neuraminic acid (Neu5Ac9Lt), 5-N-acetyl-8-O-methyl-neuraminic acid (Neu5Ac8Me) and the 1,7-lactone (Neu5Ac1,7L) of neuraminic acid. Neu4,5Ac2, relatively abundant in the gut, was present as a minor compound in all tissues, including liver, olfactory lobe, telencephalon, metencephalon and hippocampus. Neu5,9Ac2 was also found in these tissues, except in the liver. It is suggested that these sialic acids represent the endogenous substrate and receptor determinants for murine coronaviruses.

Regulation of sialic acid O-acetylation in human colon mucosa

Abstract The expression of O-acetylated sialic acids in human colonic mucins is developmentally regulated, and a reduction of O-acetylation has been found to be associated with the early stages of colorectal cancer. Despite this, however, little is known about the enzymatic process of sialic acid O-acetylation in human colonic mucosa. Recently, we have reported on a human colon sialate 7(9)-O-acetyltransferase capable of incorporating acetyl groups into sialic acids at the nucleotidesugar level [Shen et al., Biol. Chem. 383 (2002), 307317]. In this report, we show that the CMP-N-acetyl-neuraminic acid (CMPNeu5Ac) and acetyl-CoA (AcCoA) transporters are critical components for the O-acetylation of CMPNeu5Ac in Golgi lumen, with specific inhibition of either transporter leading to a reduction in the formation of CMP-5-Nacetyl-9-O-acetylneuraminic acid (CMP-Neu5,9Ac2). Moreover, the finding that 5-Nacetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) could be transferred from neo-synthesised CMP-Neu5,9Ac2 to endogenous glycoproteins in the same Golgi vesicles, together with the observation that asialofetuin and asialo-human colon mucin are much better acceptors for Neu5,9Ac2 than asialo-bovine submandibular gland mucin, suggests that a sialyltransferase exists that preferentially utilises CMPNeu5,9Ac2 as the donor substrate, transferring Neu5,9Ac2 to terminal Galβ1,3(4)R- residues.

Sialic acid metabolic engineering: a potential strategy for the neuroblastoma therapy.

Background Sialic acids (Sia) represent negative-charged terminal sugars on most glycoproteins and glycolipids on the cell surface of vertebrates. Aberrant expression of tumor associated sialylated carbohydrate epitopes significantly increases during onset of cancer. Since Sia contribute towards cell migration ( =  metastasis) and to chemo- and radiation resistance. Modulation of cellular Sia concentration and composition poses a challenge especially for neuroblastoma therapy, due to the high heterogeneity and therapeutic resistance of these cells. Here we propose that Metabolic Sia Engineering (MSE) is an effective strategy to reduce neuroblastoma progression and metastasis. Methods Human neuroblastoma SH-SY5Y cells were treated with synthetic Sia precursors N-propanoyl mannosamine (ManNProp) or N-pentanoyl mannosamine (ManNPent). Total and Polysialic acids (PolySia) were investigated by high performance liquid chromatography. Cell surface polySia were examined by flow-cytometry. Sia precursors treated cells were examined for the migration, invasion and sensitivity towards anticancer drugs and radiation treatment. Results Treatment of SH-SY5Y cells with ManNProp or ManNPent (referred as MSE) reduced their cell surface sialylation significantly. We found complete absence of polysialylation after treatment of SH-SY5Y cells with ManNPent. Loss of polysialylation results in a reduction of migration and invasion ability of these cells. Furthermore, radiation of Sia-engineered cells completely abolished their migration. In addition, MSE increases the cytotoxicity of anti-cancer drugs, such as 5-fluorouracil or cisplatin. Conclusions Metabolic Sia Engineering (MSE) of neuroblastoma cells using modified Sia precursors reduces their sialylation, metastatic potential and increases their sensitivity towards radiation or chemotherapeutics. Therefore, MSE may serve as an effective method to treat neuroblastoma.

A Novel Approach to Decrease Sialic Acid Expression in Cells by a C-3-modified N-Acetylmannosamine

Background: Inhibitors of cellular sialic acid expression offer substantial therapeutic promise for diseases associated with oversialylation. Results: 2-Acetylamino-2-deoxy-3-O-methyl-d-mannose reduces the sialic acid concentration in cells and inhibits the UDP-GlcNAc-2-epimerase/ManNAc kinase. Conclusion: Inhibition of the key enzyme of sialic acid biosynthesis by a ManNAc analog decreases cellular sialic acid expression. Significance: ManNAc analogs represent a new class of sialic acid inhibitors. Due to its position at the outermost of glycans, sialic acid is involved in a myriad of physiological and pathophysiological cell functions such as host-pathogen interactions, immune regulation, and tumor evasion. Inhibitors of cell surface sialylation could be a useful tool in cancer, immune, antibiotic, or antiviral therapy. In this work, four different C-3 modified N-acetylmannosamine analogs were tested as potential inhibitors of cell surface sialylation. Peracetylated 2-acetylamino-2-deoxy-3-O-methyl-d-mannose decreases cell surface sialylation in Jurkat cells in a dose-dependent manner up to 80%, quantified by flow cytometry and enzyme-linked lectin assays. High-performance liquid chromatography experiments revealed that not only the concentration of membrane bound but also of cytosolic sialic acid is reduced in treated cells. We have strong evidence that the observed reduction of sialic acid expression in cells is caused by the inhibition of the bifunctional enzyme UDP-GlcNAc-2-epimerase/ManNAc kinase. 2-Acetylamino-2-deoxy-3-O-methyl-d-mannose inhibits the human ManNAc kinase domain of the UDP-GlcNAc-2-epimerase/ManNAc kinase. Binding kinetics of the inhibitor and human N-acetylmannosamine kinase were evaluated using surface plasmon resonance. Specificity studies with human N-acetylglucosamine kinase and hexokinase IV indicated a high specificity of 2-acetylamino-2-deoxy-3-O-methyl-d-mannose for MNK. This substance represents a novel class of inhibitors of sialic acid expression in cells, targeting the key enzyme of sialic acid de novo biosynthesis.