Christina E. Galuska

Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis

Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. However, a prerequisite for this mode of action would be a carrier-mediated import of the hydrophilic steroid sulfate molecules into specific target cells in reproductive tissues such as the testis. In the present study we detected predominant expression of the Sodium-dependent Organic Anion Transporter (SOAT), the Organic Anion Transporting Polypeptide 6A1, and the Organic Solute Carrier Partner 1 in human testis biopsies. All of these showed significantly lower or even absent mRNA expression in severe disorders of spermatogenesis (arrest at the level of spermatocytes or spermatogonia, Sertoli cell only syndrome). Only SOAT was significantly lower expressed in biopsies showing hypospermatogenesis. By use of immunohistochemistry SOAT was localized to germ cells at various stages in human testis biopsies showing normal spermatogenesis. SOAT immunoreactivity was detected in zygotene primary spermatocytes of stage V, pachytene spermatocytes of all stages (I–V), secondary spermatocytes of stage VI, and round spermatids (step 1 and step 2) in stages I and II. Furthermore, SOAT transport function for steroid sulfates was analyzed with a novel liquid chromatography tandem mass spectrometry procedure capable of profiling steroid sulfate molecules from cell lysates. With this technique, the cellular inward-directed SOAT transport was verified for the established substrates dehydroepiandrosterone sulfate and estrone-3-sulfate. Additionally, β-estradiol-3-sulfate and androstenediol-3-sulfate were identified as novel SOAT substrates.

Engineering of complex protein sialylation in plants.

Significance Sialic acid (Sia) residues are essential monosaccharides in mammals and confer multiple biological functions. Their precise generation is important for both structure–function studies and biotechnological applications. We describe a unique technology that enables the controlled generation of protein sialylation in Nicotiana benthamiana. The plant engineering approach relies on a combination of endogenous glycan deconstruction and the introduction of human sialylation capabilities. An arrangement of transgenic and transient expression modules resulted in the targeted synthesis of Sia structures in three different linkage types, reaching a polymerization degree exceeding 40 residues (polySia). Importantly, the obtained functional activities of polySia point to novel biotherapeutic applications. Our results highlight the exceptional flexibility of the plant-based expression platform for engineering complex posttranslational protein modifications. Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions.

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines : HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.

Free and sulfated steroids secretion in postpubertal boars (Sus scrofa domestica)

Sulfated steroids have been traditionally regarded as inactive metabolites. However, they may also serve as precursors for the production of active free steroids in target cells. In this study, we used the boar as a model to study the metabolism, transport, and function of steroid sulfates due to their high production in the porcine testicular-epididymal compartment, of which the role is unknown. To characterize the secretion of free and sulfated steroids, plasma samples were collected from six postpubertal boars over 6  h every 20  min from the jugular vein. Long-term secretion profiles were also established in seven boars stimulated with human chorionic gonadotropin. To directly characterize the testicular output, samples were collected from superficial testicular arterial and venous blood vessels. Testosterone, androstenedione and sulfated pregnenolone, DHEA, estrone (E1), and estradiol-17β (E2) were determined by liquid chromatography-tandem mass spectrometry. Free E1 and E2 were measured by RIA. Irrespective of a high variability between individuals, the results suggest that i) all steroids assessed are primarily produced in the testis, ii) they exhibit similar profiles pointing to a pulsatile secretion with low frequency (three to five pulses per day), and iii) after synthesis at least a major proportion is immediately released into peripheral circulation. The fact that all steroid sulfates assessed are original testicular products and their high correlations with one another suggest their role as being intermediates of testicular steroidogenesis rather than as being inactivated end products. Moreover, a substantial use of sulfated steroids in porcine testicular steroidogenesis would assign a crucial regulatory role to steroid sulfatase, which is highly expressed in Leydig cells.

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.

Is Polysialylated NCAM Not Only a Regulator during Brain Development But also during the Formation of Other Organs

In mammals several cell adhesion molecules are involved during the pre- and postnatal development of all organ systems. A very prominent member of this family is the neural cell adhesion molecule (NCAM). Interestingly, NCAM can be a target for a special form of posttranslational modification: polysialylation. Whereas nearly all extracellular proteins bear mono-sialic acid residues, only a very small group can be polysialylated. Polysialic acid is a highly negatively-charged sugar polymer and can comprise more than 90 sialic acid residues in postnatal mouse brains increasing dramatically the hydrodynamic radius of their carriers. Thus, adhesion and communication processes on cell surfaces are strongly influenced allowing, e.g., the migration of neuronal progenitor cells. In the developing brain the essential role of polysialylated NCAM has been demonstrated in many studies. In comparison to the neuronal system, however, during the formation of other organs the impact of the polysialylated form of NCAM is not well characterized and the number of studies is limited so far. This review summarizes these observations and discusses possible roles of polysialylated NCAM during the development of organs other than the brain.

In vitro generation of polysialylated cervical mucins by bacterial polysialyltransferases to counteract cytotoxicity of extracellular histones

Neutrophil extracellular traps (NET) are formed against pathogens. However, various diseases are directly linked to this meshwork of DNA. The cytotoxic properties of extracellular histones especially seem to be an important trigger during these diseases. Furthermore, NET accumulation on implants is discussed to result in an impaired efficiency or failure, depending on the category of implant. Interestingly, mucins have been investigated as surface coatings potentially capable of reducing neutrophil adhesion. Similarly, polysialic acid was shown to inactivate the cytotoxic properties of extracellular histones. We wanted to combine the probability to decrease the adhesion of neutrophils using mucins with the capability of sialic acid polymers to counteract histone‐mediated cytotoxicity. To this end, we elongate cervical mucins using bacterial polysialyltransferases. Subsequent cell‐based experiments demonstrated the activity of elongated mucins against histone‐mediated cytotoxicity. Thus, polysialylated mucins may represent a novel component to coat implants or to combat diseases with exaggerated NET formation.

Artificial Polysialic Acid Chains as Sialidase-Resistant Molecular-Anchors to Accumulate Particles on Neutrophil Extracellular Traps

Neutrophils are involved in numerous immunological events. One mechanism of neutrophils to combat pathogens is the formation of neutrophil extracellular traps (NETs). Thereby, neutrophils use DNA fibers to form a meshwork of DNA and histones as well as several antimicrobial components to trap and kill invaders. However, the formation of NETs can lead to pathological conditions triggering among other things (e.g., sepsis or acute lung failure), which is mainly a consequence of the cytotoxic characteristics of accumulated extracellular histones. Interestingly, the carbohydrate polysialic acid represents a naturally occurring antagonist of the cytotoxic properties of extracellular histones. Inspired by polysialylated vesicles, we developed polysialylated nanoparticles. Since sialidases are frequently present in areas of NET formation, we protected the sensitive non-reducing end of these homopolymers. To this end, the terminal sialic acid residue of the non-reducing end was oxidized and directly coupled to nanoparticles. The covalently linked sialidase-resistant polysialic acid chains are still able to neutralize histone-mediated cytotoxicity and to initiate binding of these polysialylated particles to NET filaments. Furthermore, polysialylated fluorescent microspheres can be used as a bioanalytical tool to stain NET fibers. Thus, polySia chains might not only be a useful agent to reduce histone-mediated cytotoxicity but also an anchor to accumulate nanoparticles loaded with active substances in areas of NET formation.

Desialylation of spermatozoa and epithelial cell glycocalyx is a consequence of bacterial infection of the epididymis

Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in humans. In men, pathogens can also spread to the genital tract via the continuous ductal system, eliciting bacterial prostatitis and/or epididymo-orchitis. Antibiotic treatment usually clears pathogens in acute epididymitis; however, the fertility of patients can be permanently impaired. Because a premature acrosome reaction was observed in an UPEC epididymitis mouse model, and sialidases on the sperm surface are considered to be activated via proteases of the acrosome, we aimed to investigate whether alterations of the sialome of epididymal spermatozoa and surrounding epithelial cells occur during UPEC infection. In UPEC-elicited acute epididymitis in mice, a substantial loss of N-acetylneuraminic acid residues was detected in epididymal spermatozoa and epithelial cells using combined laser microdissection/HPLC-ESI-MS analysis. In support, a substantial reduction of sialic acid residues bound to the surface of spermatozoa was documented in men with a recent history of E. coli-associated epididymitis. In vitro, such an UPEC induced N-acetylneuraminic acid release from human spermatozoa was effectively counteracted by a sialidase inhibitor. These findings strongly suggest a substantial remodeling of the glycocalyx of spermatozoa and epididymal epithelial cells by endogenous sialidases after a premature acrosome reaction during acute epididymitis.

Mass Spectrometric Analysis of Oligo- and Polysialic Acids

Oligo- and polysialic acids (oligo/polySia) are involved in numerous biological processes depending on the chain length, the comprised type of sialic acids, as well as the glycosidic linkages. Here, we describe the determination of the composition, the sequence, as well as the linkages between the sialic acid residues of lactonized oligo/polySia using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)(/MS) and electrospray-ionization (ESI)-MS((n)).