MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

Imtiaj Hasan,Yasuhiro Koide,Sarkar Kawsar,Shigeki Sugawara,Jiharu Hamako,Hideho Uchiyama,Nobuhiko Kojima,Daiki Terada,Yuki Fujii,Taei Matsui,Masahiro Hosono,Yukiko Ogawa,Toshiyuki Fujiwara,Yasuhiro Ozeki,Sultana Rajia,Keisuke Takahashi,Naoya Iimura,Hideaki Fujita,Robert Kanaly

doi:10.3390/md13127071

Abstract

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

Highlights

During the present decade, genome databases have been established for numerous mollusk species, and are very useful in industrial and ecological studies [1]
Deduced a.a. from cDNA coding a Gal/GalNAc-binding lectin isolated from another mytilid mussel, Crenomytilus grayanus, gives similar primary structure [16,17]
A role of native MytiLec in supporting innate immunity could explain its cytotoxic activity against Burkitt's lymphoma cells, since the mussel and vertebrate cells may have common surface glycans such as Gb3, as well as similar cell regulatory mechanisms

Summary

Introduction

Genome databases have been established for numerous mollusk species, and are very useful in industrial and ecological studies [1]. Recent studies have focused on such genes that are capable of being activated by various environmental pollutants. Progress in this area will have important ecological, economic, and human health implications. MytiLec is an α-Gal-binding lectin that we isolated in 2012 from the Mediterranean mussel Mytilus galloprovincialis (family Mytilidae). Deduced a.a. from cDNA coding a Gal/GalNAc-binding lectin isolated from another mytilid mussel, Crenomytilus grayanus, gives similar primary structure [16,17]. In addition to possessing this characteristic folding, MytiLec was a non-covalently bound dimer consisting of two polypeptides having the glycan-binding activity in each sub-domain. MMyyttiiLLeecc ddooeess nnoott hhaavvee aaddddiittiioonnaall ffuunnccttiioonnaall ddoommaaiinnss oorr ssuubbuunniittss bbeessiiddee ggllyyccaann‐-bbiinnddiinngg ddoommaaiinnss,, iinn ccoonnttrraasstt ttoo ootthheerr RR-‐ttyyppee lleeccttiinnss,, aalltthhoouugghh iitt iiss ccaappaabbllee ooff iinndduucciinngg ccyyttoottooxxiicciittyy. TThhee mmeecchhaanniissmmss wwhheerreebbyy MMyyttiiLLeecc ttrraannssmmiittss iittss ssiiggnnaallsstthhrroouugghhcceelllslstotoacatcitviavtaetevavrairoiuosussigsinganlatlratrnasndsudcuticotniomn omleocluecleuslefsorfoinrdinudctuiocntioonf ocafnccaenrcceerllcaeplloapptoospistoasries oafregroefatgirnetaetreinstt.eWreset.useWdeexupseedrimexepnetarilmceelnl tlainl ec,eRllamlinoes, wRiatmh hosigwh litehvehlsigohf lGevb3elsexopfrGesbs3ioenxptoresstsuiodny taopsotputdoysisa‐pinodputocsinisg-inmdoulceicnuglems o(mleictuolgeesn(‐macittiovgaetend-apctriovtaetiendkpinraosteesin(MkiAnaPsKes) (cMasAcaPdKe), cmasictoacdheo, nmdirtoiac‐hcoonndtrroiall-icnogntcraoslplinasgec, aasnpdased,eaanthd dreeacethptroercespitgonrasl)ignacatli)vaactteidvabteyd MbyyMtiLyetciLeinc iBnuBrkuirtkt’isttl’ysmlypmhpohmoamcaelclse.lls

Glycan‐-Binding and Cell Agglutination of Recombinant MytiLec

Cell Lines and Culture

Cytotoxicity and Cell Viability Assays

Findings

Conclusions