The Journal of Biochemistry (JB), which celebrated its 100th anniversary this year, and our mentor, the late Dr Sen-itiroh Hakomori, shared a deep and long-lasting relationship. In 1959, Dr Hakomori moved to the Cancer Research Institute (CRI) of Tohoku Pharmaceutical University, together with his mentor, Dr Hajime Masamune. Regrettably, Prof. Masamune passed away suddenly and Dr Hakomori consequently became the head of the laboratory as a Professor at the age of 30 years. JB was the first English-language biochemistry journal to publish an article in which Dr Hakomori was listed as a corresponding author (1).
During his 4 years in this laboratory, his work shifted gradually from cancerous changes in mucoproteins to those in glycolipids, following the trend of Dr Masamune’s laboratory. He published seven papers in JB before leaving Sendai. The seventh paper described complete methylation of sugars, a technique that subsequently became widely known as the ‘Hakomori method’ (2). The method is based on a reaction of methylsulfinyl carbanions produced by dimethylsulfoxide and sodium hydride with sugars. It is a highly innovative method that makes possible complete methylation of sodium alkoxide intermediates, including difficult-to-react acidic sugars, by methyl iodide, in high yield, in a single reaction that does not require specialized equipment, and has been widely applied in glycan structural analysis. The number of citations of this paper on Web of Science to date is 5273.
The publication of this paper involved an interesting episode, described in Dr Hakomori’s essay in Current Contents in 1980 (3). He initially submitted a ‘Hakomori method’ manuscript as a full paper to JB in June 1963—and it was rejected! Dr Tamio Yamakawa, a member of the JB editorial board at the time, strongly urged him to resubmit, commenting that ‘this method was efficient enough at least for glycolipids…’ Accordingly, Dr Hakomori resubmitted the manuscript as a Letter—and it was accepted! Without the brilliant insight and forceful encouragement of Dr Yamakawa as an authority in the glycolipid field, the ‘Hakomori method’ might have been handed over to other researchers in foreign countries. This episode evokes for us the kind of transcendent ‘fate’ that we frequently observe in the natural sciences.
In the late 1960s, Dr Hakomori published a paper on the phenomenon of cancer glycosylation defects in glycolipids (reduced levels in malignant cells of glycolipids possessing elongated glycans and corresponding accumulation of their precursors) (4, 5). He also developed a method for labelling cell surface glycans with isotopes (6), which led to discovery of increased content in malignant cells of glycolipids such as paragloboside and Forssman antigen and characterization of fibronectin, a cell surface protein that shows reduced expression in cancer cells (7).
Beginning in the late 1970s, Dr Hakomori and his research colleagues focused increasingly on monoclonal antibodies (MoAbs), which had only recently been developed, and established a large variety of MoAbs that targeted glycolipids showing cancer-related changes. Among these was FH6 (8), which recognizes (sialyl Lex-i), a marker of lung adenocarcinoma, and has subsequently been applied widely for antibody-based diagnosis of cancer. Many of the group’s studies during the 1980s involved targeting of cancer-specific carbohydrate antigens (sialyl Tn, GM2, GD2, globo-H, etc.) for cancer treatment as well as diagnosis and provided a basis for today’s chemotherapeutic strategies using molecular targeted drugs. Dr Hakomori was also actively involved in the mechanisms of human blood groups, which are classical representatives of classically known cell surface glycan structures. In 1990, he and his colleague Dr Fumi Yamamoto published a landmark, much-cited paper in Nature titled ‘Molecular genetic basis of the histo-blood group ABO system’ (9).
We would like to describe here the relationship between Dr Hakomori and our laboratory, CRI, and its successor (following Dr Hakomori’s move to the USA), Institute of Molecular Biomembrane and Glycobiology (IMBG). The ‘Ganken Daiichi-bu’ of CRI, which was organized by Dr Hakomori, was overseen by his successor, Dr Hiroaki Kawauchi. Even in the USA, Dr Hakomori served as a supervisor and continued to advise Dr Kawauchi on research policies. Dr Kawauchi’s research focused mainly on animal lectins directed to cancer cells. Notably, he discovered that a novel lectin isolated from Japanese bullfrog (Rana catesbeiana) eggs agglutinated cancer cells and that its target sugar chain contained sialic acid. Around the same time, Dr Hakomori was investigating oncogenic changes of gangliosides (sialic acid-containing glycosphingolipids). He therefore became interested in the novel lectin and significantly promoted research progress in our laboratory by providing us with essential membrane glycolipid samples and encouraging us to submit to several major journals (10–12). The lectin was later characterized as a sialic acid-binding lectin (cSBL) that also displayed RNase activity. It has been passed on to the present-day Division of Cell Recognition within IMBG, whose theme is ‘Elucidation of the mechanism of action of RNase-based lectins with malignant cell-selective cytotoxicity’ (13).
Dr Hakomori’s overall research theme was ‘structure and function of glycolipids and glycoproteins as membrane components’, and his ultimate goal was to clarify ‘details of cell–cell interactions’ and ‘mechanisms of cell canceration’. The keywords ‘cell recognition’, ‘cell adhesion’ and ‘signal transduction’ are appropriate in this context. In 1990, sialyl Lex was identified as the glycan ligand on the leukocyte side of E-selectin in the lymphocyte homing phenomenon (14). Selectins are type I transmembrane proteins with a C-type lectin domain at the extracellular N-terminus, which is capable of carbohydrate binding. This is just one of several types of cell–cell interaction whereby cells bind to each other by carbohydrate–protein interaction (CPI). Identification of ligand glycans for the three types of selectins has involved many research groups and some twists and turns, but the topic has inevitably attracted considerable attention because such interactions are directly related to the mechanism of cancer metastasis. Other examples of CPI have been reported in which proteins directly recognize glycans and affect cellular functions, including those involving lectins such as galectins and sialic acid-binding immunoglobulin-type lectins (Siglecs), and binding of integrins or growth factor receptors to glycolipids (15). It has also been demonstrated that cell–cell junctions can be mediated by homophilic (e.g. Lex-to-Lex) or heterophilic (e.g. GM3-to-Gg3) interactions between glycans, which play key roles in subsequent signal transduction (16). We remember that ‘carbohydrate–carbohydrate interaction’ was a favourite phrase of Dr Hakomori that he used frequently. He published a review article that strongly emphasized this concept in JB with Dr Yasuyuki Igarashi (now at Hokkaido University), who was one of his collaborators at The Biomembrane Institute at that time (17). In 1997, Dr Kai Simons proposed a cell membrane model termed ‘lipid rafts’, which attracted attention as a signalling platform, and fit well with the previously established (1972) Singer–Nicolson ‘fluid membrane mosaic’ model. Dr Simons’ concept spread worldwide, visualized as ‘moving rafts’ drifting in a sea of lipids. Dr Hakomori, on the other hand, focused on the assembly state (organization) of glycolipid components in cell membranes (18) and developed a concept of lipid microdomains. He commented that ‘there are microdomains that do not move’ and ‘Rafts are the antithesis of the caveolae thesis. We should seek a new thesis. The ensuing glycosynapse concept’ (19), based on lipid microdomains, was based on a huge amount of evidence from Dr Hakomori’s studies over the years. Under this concept, plasma membrane microdomains are a signalling platform that controls cell adhesion, motility and growth without moving (in contrast to rafts)—a ‘new thesis’.
In the Division of Glycopathology at IMBG, we have pursued studies of glycolipid microdomain involvement in pathogenesis and proposed a working hypothesis that insulin resistance is a ‘membrane microdomain disorder’, based on findings that insulin receptors in obese adipose tissues differ from caveolae microdomains because of increased levels of ganglioside GM3 (20). We also demonstrated that a-series gangliosides in helper T (CD4+ T) cells and o-series gangliosides in killer T (CD8+ T) cells form distinct microdomains that are essential for their respective T-cell receptor-dependent T-cell subset-selective and -specific activation. These conclusions have received considerable attention and comments in the glycoimmunology field (21).
On November 10, 2017, we organized a ‘Glycoscience Symposium with Gratitude to Dr Sen-itiroh Hakomori’ to celebrate his 88th birthday (Beiju;). Dr Hakomori delivered a special lecture entitled ‘Recollection of Glycan Research and Prospects for the Future’. During the lecture, he emphasized several important topics for future investigation: (1) search for the existence of new recognition and binding modes of sugar chains, (2) common patterns of integration of sugar chains into cell membranes, (3) relationships between gene-controlled sugar chain expression patterns and diseases, (4) epigenetic control mechanisms of sugar chain expression and (5) possible phenotypic control of stem cell by sugar chains. He mentioned that we, glycoscientists, particularly those pursuing cancer-related subjects, should keep in mind and learn from Cornheim’s mystery theory: ‘cells that should become cancer in the future (cancer stem cells) are created during ontogeny and lurking in tissues, but sometimes triggers will bring out the original ability of cancer cells (Cohnheim J. 1875, Virchows Arch Pathol Anat Physiol Klin Med 65: 64)’. Intriguing topics for future research are as follows: the mechanism whereby cancer stem cells are able to ‘lurk’ in tissues, the mechanism that governs the original cancer function and the involvement of sugar chains in these mechanisms. Dr Hakomori expected that the research field of ‘tissue/organ glycobiology’, including the influence of surrounding cells on tissues and organs, will become increasingly active as it branches off from conventional ‘cell glycobiology’. We were all so impressed to his witness and passion for knowledge, like that of a young man, and his continuing dedication to research.
Professor Sen-itiroh Hakomori (1929–2020): A memorial photo taken at The Institute of Molecular Biomembrane and Glycobiology (IMBG), Tohoku Medical and Pharmaceutical University (TMPU)
