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

Abstract

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.

Sweet Modifications Modulate Plant Development

“…Both enzymes are active on N-glycans from the cell wall. α-MAN is involved in cell wall glycan trimming and β-NAHase creates paucimannosidic N-glycans by removing terminal GlcNAc or GalNAc residues [ 208 ] ( Figure 2 ). Their activity and expression levels in bell pepper were proven to increase during fruit ripening.…”

Recent Developments in Deciphering the Biological Role of Plant Complex N-Glycans

“…While GnGnXF is the dominant structure on many glycoproteins ( Zeng et al, 2018 ), on a rather small number of glycoproteins, the GnGnXF N -glycan is further modified by β1,3-galactosyltransferase (GALT1) and α1,4-fucosyltransferase (FUT13) to generate Lewis A-type structures ( Strasser et al, 2007a ; Beihammer et al, 2021 ). In the vacuole, at the plasma membrane or in the apoplast, different β-hexosaminidases can cleave off terminal GlcNAc residues from exposed complex N -glycans resulting in the formation of truncated or paucimannosidic N -glycans ( Strasser et al, 2007b ; Alvisi et al, 2021 ). The biosynthetic pathway and involved enzymes are well characterized and have been reviewed in detail recently ( Strasser et al, 2021 ).…”

Glyco-Engineering Plants to Produce Helminth Glycoproteins as Prospective Biopharmaceuticals: Recent Advances, Challenges and Future Prospects

“…Similar to the synthesis of galactose-extended N-glycans, it seems that fucosylation of LDN, to obtain LDN-F, enhances the extension of N-glycans with GalNAc (Wilbers et al, 2017). Plant-native β-hexosaminidases (HEXOs) activity may limit the generation of LDN glycan motifs on kappa-5 in GalNAcTexpressing Nicotiana benthamiana (Alvisi et al, 2021). HEXOs are hydrolytic enzymes that are able to remove terminal GlcNAc and GalNAc residues from complex N-glycans, thereby generating typical plant paucimannosidic N-glycans (Shin et al, 2017;Alvisi et al, 2021).…”

“…Plant-native β-hexosaminidases (HEXOs) activity may limit the generation of LDN glycan motifs on kappa-5 in GalNAcTexpressing Nicotiana benthamiana (Alvisi et al, 2021). HEXOs are hydrolytic enzymes that are able to remove terminal GlcNAc and GalNAc residues from complex N-glycans, thereby generating typical plant paucimannosidic N-glycans (Shin et al, 2017;Alvisi et al, 2021). Plants harbor several HEXO homologs (HEXO1, HEXO2 and HEXO3) that differ by their substrate specificity and sub-cellular localization.…”

“…N-glycans with nonendogenous GalNAc extended N-glycans can be processed by HEXO3 in the apoplast of N. benthamiana. In addition, HEXO2 was characterized as a membrane-bound enzyme specifically cleaving GalNAc residues from N-glycans (Alvisi et al, 2021). Unfortunately, targeted transient RNAi was not sufficient to improve LDN synthesis in plants (Alvisi et al, 2021).…”