Data_Sheet_1_β-Hexosaminidases Along the Secretory Pathway of Nicotiana benthamiana Have Distinct Specificities Toward Engineered Helminth N-Glycans on Recombinant Glycoproteins.pdf

Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient “helminthization” of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.

寄生虫（蠕虫）的分泌物中含有丰富多样的免疫调节性糖蛋白，这些糖蛋白具有治疗炎症性疾病，如自身免疫病的潜力。然而，由于原生寄生虫衍生蛋白的有限可获得性，识别可开发成新型生物制药的单个分子受到了阻碍。近期，开创性的研究显示，在烟草原生境植物（Nicotiana benthamiana）中可以暂时性生产蠕虫糖蛋白，同时通过共表达所需的糖基转移酶模拟其原生蠕虫N-聚糖组成。然而，通过糖基工程在植物中实现N-聚糖的有效“蠕虫化”似乎受到了β-N-乙酰己糖胺酶对复杂N-聚糖不期望的截断的阻碍，尤其是在合成具有天线GalNAcβ1-4GlcNAc（LacdiNAc或LDN）结构的N-聚糖时。在本研究中，我们从N. benthamiana中克隆了新的β-己糖胺酶开放阅读框，并表征了这些酶的生化活性。我们确定了HEXO2和HEXO3为负责切割模型蠕虫糖蛋白κ-5上天线GalNAc残基的酶。此外，我们揭示了HEXO家族的每个成员对N-聚糖底物具有独特的特异性，其中HEXO2具有严格的β-半乳糖胺酶活性，而HEXO3则切割GlcNAc和GalNAc。将HEXO2和HEXO3识别为LDN切割的主要靶点，将使针对这些N-聚糖的不期望处理进行靶向基因组编辑的方法成为可能。这些酶的有效敲除将允许在植物中生产具有定制蠕虫N-聚糖的、具有治疗相关性的糖蛋白。