“…The resulting S2 cell derivatives produced endogenous and recombinant glycoproteins with significantly lower levels of paucimannosidic and higher levels of elongated N-glycan structures 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 (Supporting Information Table S2). In fact, the quantitative extent of the structural changes obtained using CRISPR-Cas9 editing far exceeded the extent of those observed in several previous reports in which RNA interference was used to try to modify insect cell N-glycan processing pathways by repressing fdl gene expression 39,[42][43][44] . An important caveat, however, is that these studies were done in different systems under different culture conditions.…”
Section: Impact Of Fdl Editing On N-glycan Processing In Insect Cellsmentioning
Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect N-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the N-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man3GlcNAc2), and led to the production of partially elongated, mammalian-type complex N-glycans (GlcNAc2Man3GlcNAc2) in S2 cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit insect cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing insect and mammalian N-glycan processing pathways. Finally, the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures.
“…The resulting S2 cell derivatives produced endogenous and recombinant glycoproteins with significantly lower levels of paucimannosidic and higher levels of elongated N-glycan structures 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 (Supporting Information Table S2). In fact, the quantitative extent of the structural changes obtained using CRISPR-Cas9 editing far exceeded the extent of those observed in several previous reports in which RNA interference was used to try to modify insect cell N-glycan processing pathways by repressing fdl gene expression 39,[42][43][44] . An important caveat, however, is that these studies were done in different systems under different culture conditions.…”
Section: Impact Of Fdl Editing On N-glycan Processing In Insect Cellsmentioning
Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect N-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the N-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man3GlcNAc2), and led to the production of partially elongated, mammalian-type complex N-glycans (GlcNAc2Man3GlcNAc2) in S2 cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit insect cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing insect and mammalian N-glycan processing pathways. Finally, the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures.
“…Insights into the glycosylation machinery of other insect species were achieved by profiling cell lines of the cabbage moth ( Mamestra brassicae ) (Mb0503), silkworm ( Bombyx mori ) (Bm‐N), fall armyworm ( Spodoptera frugiperda ) (Sf‐9 and Sf‐21), the cabbage looper ( Trichoplusia ni ) (Tn‐4h and BTI‐TN5B1‐4 known as High Five cells), and the gypsy moth ( Lymantria dispar ) (Ld652Y) (Kubelka et al ., ; Hsu et al ., ; Takahashi et al ., ; Choi et al ., ; Misaki et al ., ; Nomura et al ., ; Stanton et al ., ). The glycoprofiling demonstrated that PMGs are highly abundant N ‐glycans in these insect cell lines (∼40% of the N ‐glycome) (see Table S1).…”
Section: Surveying Pmps Across the Eukaryotic Kingdoms And Phylamentioning
confidence: 99%
“…Insect cell lines have been frequently utilised for recombinant expression of mammalian glycoproteins. Mouse interferon‐β (Misaki et al ., ), human IgG1 (Park et al ., ) and calf alkaline phosphatase (ALP) (Nomura et al ., ) are examples of recombinant mammalian glycoproteins expressed in silkworm cell lines decorated with common PMGs (Glycans #2a/b, #4, #10a/b, #12, and #28). Recently, B. mori was glyco‐engineered to express human N ‐acetylglucosaminyltransferase II (MGAT2) and bovine β1,4‐galactosyltransferase I (B4GALT1) to generate recombinant proteins with mammalian‐like glycosylation features in the posterior silk glands of the silkworm.…”
Section: Surveying Pmps Across the Eukaryotic Kingdoms And Phylamentioning
Paucimannosidic proteins (PMPs) are bioactive glycoproteins carrying truncated α-or β-mannosyl-terminating asparagine (N )-linked glycans widely reported across the eukaryotic domain. Our understanding of human PMPs remains limited, despite findings documenting their existence and association with human disease glycobiology. This review comprehensively surveys the structures, biosynthetic routes and functions of PMPs across the eukaryotic kingdoms with the aim of synthesising an improved understanding on the role of protein paucimannosylation in human health and diseases. Convincing biochemical, glycoanalytical and biological data detail a vast structural heterogeneity and fascinating tissue-and subcellular-specific expression of PMPs within invertebrates and plants, often comprising multi-α1,3/6-fucosylation and β1,2-xylosylation amongst other glycan modifications and non-glycan substitutions e.g. O-methylation. Vertebrates and protists express less-heterogeneous PMPs typically only comprising variable core fucosylation of bi-and trimannosylchitobiose core glycans. In particular, the Manα1,6Manβ1,4GlcNAc(α1,6Fuc)β1,4GlcNAcβAsn glycan (M2F) decorates various human neutrophil proteins reportedly displaying bioactivity and structural integrity demonstrating that they are not degradation products. Less-truncated paucimannosidic glycans (e.g. M3F) are characteristic glycosylation features of proteins expressed by human cancer and stem cells. Concertedly, these observations suggest the involvement of human PMPs in processes related to innate immunity, tumorigenesis and cellular differentiation. The absence of human PMPs in diverse bodily fluids studied under many (patho)physiological conditions suggests extravascular residence and points to localised functions of PMPs in peripheral tissues. Absence of PMPs in Fungi indicates that paucimannosylation is common, but not universally conserved, in eukaryotes. Relative to human PMPs, the expression of PMPs in plants, invertebrates and protists is more tissue-wide and constitutive yet, similar to their human counterparts, PMP expression remains regulated by the physiology of the producing organism and PMPs evidently serve essential functions in development, cell-cell communication and host-pathogen/symbiont interactions. In most PMP-producing organisms, including humans, the N -acetyl-β-hexosaminidase isoenzymes and linkage-specific α-mannosidases are glycoside hydrolases critical for generating PMPs via N -acetylglucosaminyltransferase I (GnT-I)-dependent and GnT-I-independent truncation pathways. However, the identity and structure of many species-specific PMPs in eukaryotes, their biosynthetic routes, strong tissue-and development-specific expression, and diverse functions are still elusive. Deep exploration of these PMP features involving, for example, the characterisation of endogenous PMP-recognising lectins across a variety of healthy and N -acetyl-β-hexosaminidase-deficient human tissue types and identification of microbial adhesins reactive to human PMPs, are amongs...
“…Pauci- and oligomannosidic glycans were often produced on recombinant hIgG expressed in silkworm pupae without the coexpression of human glycosyltransferases. This N -glycan is sometimes observed in expressed recombinant proteins in insects 6, 19, 31 . This result suggests that silkworm pupae are capable of producing complex N -glycans on recombinant hIgG, albeit at somewhat lower levels than those in other systems.Figure 6Chromatograms of PA-derivatized N -glycans derived from recombinant IgG from silkworm coexpressed with hGnT II and hGalT I, as determined via ODS column chromatography.…”
Section: Resultsmentioning
confidence: 91%
“…Similarly to those in insect cells, most of the N -glycans on glycoproteins expressed in silkworms are of the pauci-mannose type 19, 20 . In the posterior silk gland (PSG), up to 17% of N -glycans are terminally galactosylated in transgenic silkworm larvae coexpressing human GnT II and bovine GalT I under the control of a PSG-specific promoter 21 .…”
Recombinant proteins produced in insect cells and insects, unlike those produced in mammalian cells, have pauci-mannose-type N-glycans. In this study, we examined complex-type N-glycans on recombinant proteins via coexpression of human β-1,2-N-acetylglucosaminyltransferase II (hGnT II) and human β1,4-galactosyltransferase (hGalT I) in silkworm pupae, by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The actin A3 promoter from B. mori and the polyhedrin promoter from Autographa californica multiple nucleopolyhedroviruses (AcMNPVs) were used to coexpress hGnT II and hGalT I. These recombinant BmNPVs were coexpressed with human IgG (hIgG), hGnT II and hGalT I in silkworm pupae. When hIgG was coexpressed with hGnT II, approximately 15% of all N-glycans were biantennary, with both arms terminally modified with N-acetylglucosamine (GlcNAc). In contrast, when hIgG was coexpressed with both hGnT II and hGalT I under the control of the polyhedrin promoter, 27% of all N-glycans were biantennary and terminally modified with GlcNAc, with up to 5% carrying one galactose and 11% carrying two. The obtained N-glycan structure was dependent on the promoters used for coexpression of hGnT II or hGalT I. This is the first report of silkworm pupae producing a biantennary, terminally galactosylated N-glycan in a recombinant protein. These results suggest that silkworms can be used as alternatives to insect and mammalian hosts to produce recombinant glycoproteins with complex N-glycans.
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