Introduction to Galectins-Experimental Protocols and Unsolved Questions (Narratives)
Oct 01, 2020
Introduction to Galectins Experimental Protocols and Unsolved Questions (Narratives) （Glycoforum. 2020 Vol.23 (5), A14） DOI: https://doi.org/10.32285/glycoforum.23A14
Jun Hirabayashi / Sachiko Sato
Jun Hirabayashi
National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan. Ph.D, Science
After graduated from Tohoku University (Master of Science), he started his professional carrier at Teikyo University under Prof. Kenichi Kasai for the investigation of animal lectins. On the occasion of GlycoXV (Tokyo, 1999), he proposed the concept glycome; for this realization, he moved to National Institute of Advanced Industrial Science and Technology (AIST, Tsukuba) in 2002, and was involved in a series of national projects for glycan engineering, while he was a deputy director in Research Center for Medical Glycoscience (2006~), prime senior researcher of Research Center for Stem Cell Engineering (2012~). Now, he is a distinguished senior researcher in AIST, while being a vice president of the Japanese Society of Carbohydrate Research (JSCR) and Japanese Consortium for Glycoscience and Glycotechnology. (JCGG). He is also a visiting professor of Kagawa University (2003~) and Yokohama City University (2019~).
Sachiko Sato
Research Centre for Infectious Diseases, Faculty of Medicine, Laval University, Quebec City, Canada. Ph.D, Pharmaceutical science
Sachiko Sato graduated from Faculty of Pharmaceutical Science, Chiba University. She joined as postgraduate student in the laboratory of Dr. Akira Kobata, the Institute of Medical Science, the University of Tokyo, Japan in 1987. She also worked in the laboratory of Dr. R. Colin Hughes, MRC: National Institute for Medical Research in London, UK, where she first encountered a cytosolic mammalian lectin, now called galectin-3. She obtained her Ph. D. from the University of Tokyo in 1994. As postdoctoral fellow in the laboratory of Dr. Ron Kopito, Stanford University, she was involved in the work on cystic fibrosis. She became principal investigator of the laboratory of glycobiology in Research Center for Infectious Diseases, and assistant professor of the Faculty of Medicine, Laval University, Quebec, Canada in 1999 and is full professor since 2010. She is also director of the Bioimaging platform since 2003.
A brand new Glycoforum® genre series related to Galectins will be started, combined with publication of many detailed experimental protocols for galectin production. As editors of this series we hope to bring unique perspectives to the field of galectins and glycoscience.
Galectins are defined as an evolutionarily related family of proteins (Pfam ID: PF00337) with conserved amino acid sequences and sugar-binding specificity confined to β-galactoside 1. They are a large group of carbohydrate-binding proteins in animals and many other multicellular organisms. Galectins have been investigated over a long period (Fig. 1) and found to participate in an extensive range of biological processes, such as carcinogenesis, immunity, development, infections, etc. (see Atlas of galectin in Fig. 2; modified from the original figure from ref. 2). Despite the long history of galectin research, many unsolved mysteries remain: Prof. Kasai once referred to these mysteries as the “Seven wonders of galectins”3: namely, 1) What is the general mission of galectins? 2) Why are they extracellularly located in spite of the fact that they were designed to be intracellular proteins? 3) How do cells externalize them? 4) Why are all galectins galactoside-specific? 5) What are their extracellular and intracellular ligands? 6) Why are they localized at sites where the risk of oxidative inactivation is high? 7) Why do galectins and legume lectins have a common topology? It is simply surprising that these questions were raised almost a quarter century ago, but none of them have been answered until today.
Fig. 1. Epoch-making events in the history of galectin research. Based on the judgment of the editors (J.H. and S.S.), the focus was selected to be relatively early investigations.
* This classification of galectin names is based on the observation that the galectins studied in the early days of galectin research lost their carbohydrate-binding activity in the absence of thiol (SH)-reducing reagents, such as 2-mercaptoethanol, but this naming turned out later to be not valid for many other members (nowadays, it is understood that readily oxidatively-inactivated galectin-1 is exceptional). This confusion was one of the reasons for redefinition of the systematic name “galectin” by a group of researchers in 1994, led by SH Barondes.
References (in part of Fig. 1)
Figure 2. An atlas of galectins.
The principal locations of galectins (light pink), their ligands, complex and mucin type glycans (light blue) and oligomannose type glycans (light yellow) are shown. Galectin molecules are depicted as pink dots.
A: Cytosolic galectins can be released into the extracellular milieu through regulated secretion (a) and passive release from damaged cells (b). In the case of regulated secretion (a), galectins are known to be secreted using four pathways: transporter-, vesicle-, and autophagy-mediated and ectocytosis pathways.
B: Once released to the extracellular milieu, galectins exert many functions in a spatiotemporal and context-dependent manner. Most of those functions rely on their glycan binding activity and multivalency in their binding to glycans. Some of those functions are illustrated here.
C: Within the cytosol, galectins mediate various responses through glycan-galectin and/or protein-galectin interactions. Some of those functions are illustrated here.
For a detailed explanation, please refer to either the Glycoforum article by S. Sato or the reference (S. Sato, Mammalian cytosolic galectins act as damage-associated molecular patterns, resolutions-associated molecular patterns, and pattern-recognition receptors in innate immunity, Comprehensive Glycoscience 2nd Edition in press).
In the same paper, however, he gave a unique, witty answer to the first question, i.e., “Galectins are intelligent glue as well as non-bureaucratic bureaucrats or almighty supporting actors”. Questions 2, 3, 5, and 6 are about the binary roles of galectins in both cytoplasmic and extracellular spaces. Probably the most essential question, if combining them, is on the “origin” of galectins. To make a long history short, this is the ultimate, chicken or egg-type question; that is, “Were galectin ancestors born as lectins or non-lectin proteins?” Considering that none of the galectins identified so far have a signal sequence for secretion and none of them are glycosylated, their ancestors must have originated as cytoplasmic proteins essentially without glycans, but their function(s) as lectins in cytoplasm remained elusive until recently.
The remaining two, i.e., questions 4 and 7, are about the protein scaffold (β-sandwich/jellyroll) and carbohydrate-specificity of galectins. In other words, another essential question is “Why is galectins’ specificity confined to β-galactoside?”, but nobody seems to have found an a priori answer to it. This is again an issue of galectins evolution, but can we find any useful structural clue to answer this question?
The Galectins series consists of two parts: Part I is Experimental protocols for galectin expression. Experience-based detailed established protocols are kindly provided by Nozomu Nishi (Kagawa University, Japan). As many galectin researchers know, he has long been involved in basic researches including studies on the production of a series of galectins (mainly of human origin) and their useful mutants, including the stabilized cysteine-less Gal-1 mutant (designated CSGal-1) 4 and linker-deleted Gal-8 and Gal-9 mutants (G8Null, G9Null) 5. He also intended to deliver a wide range of expression plasmids through a public bio-resource center at RIKEN (Wako, Japan). His detailed protocol is not only for those who have been working on recombinant lectins but also for researchers including young scientists, who are not familiar with lectins purification and find the biochemical protocol currently used to produce recombinant proteins, extremely challenging, even with the fully established Escherichia coli system. It is true that there are many pitfalls, the details of which are not necessarily described in the original research papers. The provided experimental protocols consist of 1) a “27-step experimental protocol” with helpful notes, 2) “comments referring to individual human galectins”, and 3) “supplementary material” including other related experimental methods (e.g., how to concentrate galectin samples before SDS-PAGE) and specific comments (e.g., on how to purify Gal-9 under acidic conditions).
Part II is a series of Narratives, entitled “Unsolved Questions about Galectins”, which focus on some of the important unsolved questions mentioned above, from an evolutionary perspective. For this series, we invited a few world-renowned scientists who have worked on galectins or galectin-related issues for many years. They provide attractive topics, sometimes giving us bold hypotheses and exciting imaginative speculation, which would be difficult to publish in conventional scientific literature. Indeed, it is our very intention to initiate this series and by doing so, create opportunities to discuss their views and perspectives on galectins from different angles. As far as we know, no such attempt has been made, while several successful special issues on galectins have been published6-9. We greatly thank the following authors for their understanding of our planning concept and kindly accepting our invitation to write about their own interests (the order of publication may change, and additional author(s) might join in this forum). As editors of this series, we hope to bring unique perspectives into the field of galectins and glycoscience.
■ Kenichi Kasai (Teikyo University, Japan)
■ Hans-Joachim Gabius (Ludwig-Maximilians-University Munich, Germany)
■ Hakon Leffler (Lund University, Sweden)
■ James W. Dennis (Mount Sinai Hospital, Toronto, Canada)
■ Tadasu Urashima (Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan)
■ Sachiko Sato (Laval University, Quebec City, Canada)
■ Jun Hirabayashi (AIST, Tsukuba, Japan)
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Sato S (2020) Galectins and innate immunity, Comprehensive Glycoscience 2nd edition Vol 5 Chapter 147. In press
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