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Werner Reutter: A Visionary Pioneer in Molecular Glycobiology

2017; Wiley; Volume: 18; Issue: 13 Linguagem: Inglês

10.1002/cbic.201700277

1439-7633

Stephan Hinderlich, R Tauber, Carolyn R. Bertozzi, Christian P. R. Hackenberger,

Biochemical and Molecular Research

A creative pioneer: Werner Reutter (1937–2016) was a scientist who both made fundamental discoveries in glycobiology and reached out to disciplines beyond his core field. Many of his former colleagues and students will remember his desire to exchange research ideas, which ultimately contributed to the birth of new research fields. The advancement of natural life sciences is undoubtedly connected to visionary and interdisciplinary thinking. We all know and admire scientists, who have made fundamental contributions to science, which not only changed our comprehension of nature but also had a tremendous impact on other scientific disciplines unrelated to their initial discovery. The late Prof. Dr. med. Werner Reutter (1937–2016) was such a role model, a scientist who achieved both; making fundamental discoveries that advanced our understanding of the molecular principles in biological and medical research, and simultaneously reaching out to scientific disciplines outside his core expertise to change the state-of-the-art. His enthusiasm as a trained medical doctor for unravelling the molecular principles and mechanisms in glycobiology was paired with a strong interest in the chemical and physical sciences. Many of his former colleagues and students will vividly remember him for his desire to exchange research ideas that ultimately contributed to the birth of new research fields, thus making Werner Reutter one of the most influential glycobiologists to date. After having completed his studies in physics and medicine at the universities of Freiburg and Munich (Germany), Werner Reutter worked on the biosynthesis and mechanisms of action of anaphylotoxin for his doctoral thesis at the Institute of Pharmacology at Albert Ludwigs University in Freiburg. Being particularly attracted to research, he started his scientific career in 1964 with Karl Decker at the Institute of Biochemistry of the University of Freiburg, first as a Research Fellow, later as a Research Assistant (Figure 1). In 1972 he became Lecturer for Biochemistry and Pathobiochemistry. During these first years of his scientific career, Werner Reutter's work focussed on the biochemical mechanisms of liver and kidney injury, and he achieved several important research results. Among these was the observation that repeated intraperitoneal injections of d-galactosamine result within 24–48 hours in rat liver damage closely resembling human viral hepatitis.1, 2 Together with Dieter Keppler and Karl Decker (Figure 2), he showed that the liver damage results from a decrease in uridine nucleotides, impaired glycoprotein biosynthesis and reduced total protein biosynthesis. Later on, in collaboration with Chris Galanos, Werner Reutter showed that treatment of rabbits, rats and mice with d-galactosamine increased their sensitivity to the lethal effects of lipopolysaccharide several thousand fold.3 The d-galactosamine-induced experimental hepatitis and the d-galactosamine lethality model have since served as a valuable experimental system for studying liver injury,4 and to test for synergistic behaviour between different bacterial components. Werner Reutter at the farewell celebration for Norris P. Wood in 1972 (Institute of Biochemistry of the University of Freiburg) Werner Reutter (middle), Karl Decker (left) and Dieter Keppler (right) in 1984 at the “Alte Gerichtslaube” in Freiburg, Germany. Undoubtedly intrigued by these findings, Werner Reutter then turned his attention to sugar and glycoprotein metabolism, especially the metabolism of d-galactose, l-fucose and sialic acid. In this work, he developed pioneering ideas and concepts that still today deeply influence glycobiology. These achievements include the synthesis of the l-fucose amino analogue l-fucosamine.5 This research gave rise to the development of novel fucokinase inhibitors, and, as early as 1982, to the proposal of using sugar analogues as inhibitors of glycosylation. In this vein, Werner Reutter and colleagues demonstrated that the nonphysiological sugar 2-deoxy-d-galactose was incorporated into rat liver glycoconjugates and decreased the incorporation of l-fucose.6 Of particular significance were the first measurements of GDP-l-fucose,7 of fucosylated glycoproteins in liver and hepatoma,8 and of the specific activity of fucosyltransferases in liver, hepatoma and patients' sera7, 9 that resulted in the finding that the content of GDP-l-fucose, the activity of fucosyltransferases and the content of protein-bound l-fucose was strongly elevated in malignancy. On the other hand, the discrepancy of increased content of membrane-bound sialic acid accompanied with reduced intracellular biosynthesis in malignant compared to nonmalignant hepatic tissue was revealed.10 Based on these findings, Werner Reutter very quickly pointed to the particular roles l-fucose and sialic acid have in cancer, and he was among the first researchers to recognise the role of these sugars in cancer biology. This led him to ask questions about the dynamics and the role of glycoproteins in the plasma membrane. Stimulated by the novel concepts of biosynthetic oligosaccharide processing at this time, Werner Reutter turned his attention to the turnover of different sugar residues within the glycans of cell-surface glycoproteins. In vivo studies on their half-lives mainly conducted by his co-workers Wolfgang Kreisel and Rudolf Tauber showed that l-fucose and sialic acid exhibited faster turnover rates than d-mannose.11, 12 These findings gave rise to the concepts that cell-surface glycoproteins undergo remodelling in that the outer sugar residues of glycans may be partly removed by glycosidases, and that the glycoproteins may subsequently be re-fucosylated and re-sialylated during recycling from the cell surface to the trans-Golgi/trans-Golgi network, thus contributing to the dynamics of cell surface glycosylation. In 1978, Werner Reutter was appointed Associate Professor at the University of Freiburg, then in 1979, Full Professor of Biochemistry and Pathobiochemistry and Head of the Institute of Biochemistry and Molecular Biology at the Freie Universität Berlin. He continued studies on plasma membrane proteins, and in 1982 he unravelled the mystery of a “110 000-dalton glycoprotein” which was identified as dipeptidylpeptidase IV.11, 13 This work was the starting point for research on several membrane receptors in his growing research group, culminating in the Collaborative Research Center SFB 366 “Cellular Signal Recognition and Transduction”, over which Werner Reutter presided as a spokesman from 1994 to 2005 (Figure 3). However, research on membrane receptors, in particular on dipeptidylpeptidase IV, was continued after his official retirement in 2005 with a final senior-authored publication in 2012.14 Workgroup of Werner Reutter in February 2005 (Institute of Biochemistry and Molecular Biology of the Freie Universität Berlin, Berlin–Dahlem, Germany). Besides the work on membrane proteins, Werner Reutter continued his exploration of inhibitors for glycoprotein biosynthesis.15 In the 1990s, he mainly focussed on the Roseman–Warren biosynthetic pathway of sialic acids.16 These studies were accompanied by purification of the enzymes involved. In particular UDP-GlcNAc 2-epimerase and ManNAc kinase, which catalyse the first two steps of this pathway, were handled. Two separate groups of graduate and doctoral students worked on each of those enzymes over the years. In 1995, two of these co-workers, namely Roger Stäsche working on UDP-GlcNAc 2-epimerase and Stephan Hinderlich in charge of ManNAc kinase, made a vital discovery for young scientists: in ongoing experiments they observed that samples containing the two enzymes, actually supposed to be prepared independently, looked very similar in SDS-PAGE and other analyses. Obviously traumatised that one of them might have worked on the wrong enzyme for years, maybe due to the everyday mess in the laboratory and a mix-up of test tubes, they imagined the end of their scientific careers. Fortunately, the next group meeting was scheduled a few days later and some samples of almost-pure enzymes were still available. These were tested for UDP-GlcNAc 2-epimerase and ManNAc kinase activities, and each of the samples displayed activity in both assays. This was the first hint that a bifunctional enzyme might be the explanation for the results, as was confirmed by subsequent studies.17 In the group meeting, Werner Reutter was informed for the first time, that two of his PhD students had evidently been working on the same enzyme without knowing. Werner Reutter was certainly an experienced scientist, steeled by several lab catastrophes, and it was almost impossible to unsettle his positive and optimistic temperament. Nevertheless, eye witnesses reported on this day that at least a trace of shock was seen in his face; however, only a few minutes later he relaxed and immediately started to make new plans based on this fundamental finding. Research on UDP-GlcNAc 2-epimerase/ManNAc kinase, also abbreviated to GNE, speeded up not only for the Reutter group, but also for other scientists. Basic biochemical characterisation was followed by evaluation of the cellular function of the bifunctional enzyme18 and the generation of a knock-out mouse.19 All of this work revealed the key role of GNE in sialic acid biosynthesis, and confirmed Werner Reutter's anticipatory ideas of more than 20 years before. A new and unexpected input came from the group of Stella Mitrani-Rosenbaum in 2001, when they reported that mutations in the GNE gene cause a muscle disease now called GNE myopathy.20 A long-lasting and fruitful collaboration of the two groups from Israel and Germany started. In particular, Werner Reutter took to it like a duck to water in light of his background as a physician. His secretary could remember dozens of phone calls with desperate patients, longing for immediate therapy. Werner Reutter answered their questions with endless patience. More that 15 years later, an effective therapy for the disease is still not available, but Werner Reutter never lost his optimism that it would be possible in the future. The availability of recombinant GNE was very helpful for structural analysis, which in turn could be used for rationale inhibitor design in collaboration with organic chemists such as the groups of Richard Schmidt, Athanassios Giannis and Christian Hackenberger. They first focussed on UDP-GlcNAc 2-epimerase, which catalyzes the key step of sialic acid biosynthesis.21, 22 In one of Werner Reutter's late works, the crystal structure of the ManNAc kinase domain of the bifunctional enzyme with its ligands was solved; this led to the rational design of 6-O-acetyl-ManNAc23 or selenide derivatives24 as potent inhibitors. The high-throughput screening of ManNAc kinase inhibitors published in this issue25 is one of these. During the characterisation of the key enzymes involved in the biosynthesis of sialic acids, the Reutter group became interested in studying the substrate specificity of enzymes, like GNE, with unnatural mannosamine derivatives, including N-propionylmannosamine (ManNProp). In several findings covering decades of research and using numerous analytical techniques, Werner Reutter was able to show that N-acylated mannosamines were indeed transferred to asialoglycans on the cell surface.26 These findings were later applied by him and other co-workers to study biological and medicinal mechanisms such as nerve regeneration, dopamine secretion and cell stimulation to name but a few. It is probably already common knowledge in the history of modern science that Werner Reutter's findings on the substrate promiscuity of the enzymes involved in sialic acid biosynthesis, referred to as metabolic glycoengineering, were essential to the birth of a new research area in chemical biology. It was Carolyn Bertozzi who, with the aid of unnatural ketone- or azide-containing mannosamine derivatives, was able to introduce the concept of bioorthogonal chemistry for the chemical labelling of sialic acids on living cells and in live animals in several seminal papers in the late 1990s and early 2000s (Figure 4).27, 28 Years later, Werner Reutter's laboratory also contributed to this area in a collaboration with the lab of Valentin Wittmann on the use of the inverse-electron-demand Diels–Alder reaction for labelling of alkene-functionalized sialic acids.29 For further details on the topic of metabolic glycoengineering, the interested reader is referred to a very comprehensive review by Werner Reutter and co-workers written just weeks before he passed away, which was published in Angewandte Chemie.30 Carolyn Bertozzi and Werner Reutter on the occasion of her being awarded an honorary PhD at the Freie Universität Berlin in October 2014. The backstory behind this convergence of ideas paving the path towards bioorthogonal labelling highlights Werner Reutter's human gifts as a colleague and mentor. Carolyn Bertozzi was a postdoctoral fellow in Steve Rosen's lab at UCSF when she first met Werner Reutter at a conference in Southampton, England. It was an unlikely meeting. The conference was sponsored by the German and Irish Anatomical Societies and had nothing to do with glycoscience per se; moreover, Carolyn was a last-minute substitute for her postdoctoral advisor, happy to enjoy a trip to Europe and the chance to present her work on selectin ligands to a new audience. At the time, she was crafting research proposals in anticipation of faculty job applications and keenly interested in pursuing methods to image glycans on cell surfaces, ideally in live organisms. This is where the notion of bioorthogonal chemistry was rooted, but what was missing was a mechanism to introduce novel functionality into the glycans of live cells. On a cool cloudy day in Southampton that summer, Werner Reutter delivered a lecture to a largely disengaged audience of mostly older European physicians on his finding that the sialic acid biosynthetic enzymes would process unnatural N-acyl-modified mannosamine analogues. N-Propanoyl, N-butanoyl, even N-pentanoyl mannosamines, when fed to cells, were converted into the corresponding cell-surface N-acyl modified sialosides. Werner Reutter was exploring applications of this finding to modulate viral receptor interactions. But Carolyn Bertozzi wondered whether this could be a conduit for introducing bioorthogonal functionality into some of the most interesting glycans one might like to image. So, during a break, she approached him—then known to her as Prof. Dr. Reutter—to find out more about the process. And to her delight, he warmly invited her to join him for lunch where they commiserated with each other on their shared disconnection at this particular meeting. He also generously shared his detailed knowledge of the parameters of unnatural sialic acid biosynthesis and offered to help Carolyn in her pursuit of her own independent lab. A few years later, ketone- and then azide-modified mannosamine analogues populated the catalogues of several reagent suppliers and become widely used as sialoside probes. Several groups expanded the concept of bioorthogonal glycan labelling to other sugars, many model organisms, and recently to ex vivo cultured human tissues.31 Indeed, far beyond glycoscience, people interested in protein, nucleic acid and lipid labelling have also enjoyed the fruits of those early interactions with Werner Reutter. Carolyn Bertozzi will always remember him as an inspiration, a generous mentor who treated even the youngest, most naïve co-workers (such as her postdoctoral self) with kindness and respect, as colleagues and peers. When Prof. Reutter passed away in May 2016 after a short and severe illness, the scientific community lost one of its most creative, pioneering and yet humble members, who set standards in understanding the principles of glycobiology. He will certainly be missed, but his thinking and education will be maintained by his former students, colleagues, collaboration partners, competitors and scientists exploring fundamental molecular principles in glycobiology with a range of different techniques. On what would have been his 80th birthday, 5th February 2017, about one year after he passed away, a reunion was organised by his former students during which a questionnaire was given to attendees as well as colleagues worldwide asking for Werner Reutter's most outstanding scientific contributions and his most remembered character traits. Additionally, the idea was born to prepare this special issue dedicated to his life with a collection of papers from scientists and colleagues working in the greater area of glycobiology, thereby covering research that was pioneered by this seminal glycobiologist. The authors of this editorial preface as well as the editors of this special ChemBioChem issue would like to leave the reader with these excellent papers, on topics that include sialic acid biosynthesis and recognition, glycosyltransferases and metabolic oligosaccharide engineering, and finally with a quote given by one of his former colleagues from the above-mentioned questionnaire, which describes Werner Reutter as much as a short sentence can. Asked about what will be remembered of Werner Reutter, he stated: “… his great friendliness and his ability to bring together molecular and medicinal thinking” or in German “…seine große Freundlichkeit und seine Fähigkeit, immer wieder molekulare und medizinische Denkweise zusammenzuführen” Stephan Hinderlich, Rudolf Tauber, Carolyn R. Bertozzi and Christian P. R. Hackenberger