ABSTRACT The silkworm, _Bombyx mori_, is an attractive host for recombinant protein production due to its high expression efficiency, quality, and quantity. Two expression systems have been

widely used for recombinant protein production in _B. mori_: baculovirus/silkworm expression system and transgenic silkworm expression system. Both expression systems enable high protein

production, but the qualities of the resulting recombinant proteins have not been well evaluated. In this study, we expressed bovine interferon γ (IFN-γ) using the two systems and examined

the quality of the resulting proteins in terms of _N_-glycosylation and protein cleavage. Both expression systems successfully produced IFN-γ as an _N_-glycoprotein. Although the production

system, unintentional protein modification might occur, and therefore protein expression in the transgenic silkworm expression system is preferable from the point-of-view of

_N_-glycosylation of the recombinant protein and evasion of unexpected attack by a protease in _B. mori_. SIMILAR CONTENT BEING VIEWED BY OTHERS DEVELOPMENT OF A NOVEL HETEROLOGOUS GENE

EXPRESSION SYSTEM USING EARTHWORMS Article Open access 14 April 2021 ECTOPIC EXPRESSION OF SERICIN ENABLES EFFICIENT PRODUCTION OF ANCIENT SILK WITH STRUCTURAL CHANGES IN SILKWORM Article

Open access 22 October 2022 PRODUCTION OF NOROVIRUS-, ROTAVIRUS-, AND ENTEROVIRUS-LIKE PARTICLES IN INSECT CELLS IS SIMPLIFIED BY PLASMID-BASED EXPRESSION Article Open access 27 June 2024

INTRODUCTION In response to the increasing demands for protein therapeutics for human use, many biopharmaceutical protein productions have been attempted using various production hosts, and

many of the resulting products have been approved1. These biopharmaceutical protein productions have traditionally relied on bacterial fermentation or mammalian cultured systems, but they

have been hampered by various host-specific limitations with respect to scalability, production costs, contamination risk, and post-translational modifications2,3. In fact, in regard to

contamination risk, it has been reported that an accidental virus infection of mammalian cells in a manufacturing facility spoiled a bioproduction run of therapeutic proteins and ultimately

resulted in improper treatment of patients4. Therefore, there is an urgent need for improved alternative protein production platforms. The establishment of various cell lines, mainly

lepidopteran cell lines, and protein expression systems utilizing baculovirus that encode a gene of interest have enabled the use of insect cells as protein production hosts in basic

research as well as in the manufacture of biologicals for human and veterinary use5,6. However, the use of insect cells for protein production still has disadvantages in terms of maintenance

and production costs, the complexity of media, potential protein degradation caused by proteases, and the risk of mammalian virus infections3,7. Moreover, the identification of contaminant

viruses in insect cells8,9,10,11 has rendered these cells less desirable as a biopharmaceutical protein production system for human use, although two of these viruses identified flock house

virus and Sf-rhabdovirus are not considered to be mammalian pathogens5. In contrast, the silkworm _Bombyx mori_ was recently identified as an attractive insect host with promise for

recombinant proteins production. Although the _B. mori_ latent virus (BmLV) was found in _B. mori_-cultured BmN cells12, and three rhabdovirus-like sequences on _B. mori_ were identified6,

thus far, neither BmLV proliferation nor the presence of the three rhabdovirus-like viruses in larvae has been reported. These facts suggest that _B. mori_ larvae are free from virus

infection, and that recombinant protein production in _B. mori_ larvae meets the biosafety standards required for biopharmaceutical production. Owing to the establishment of two silkworm

expression systems a baculovirus expression system using _B. mori_ nucleopolyhedrovirus and silkworm larvae13,14 and a transgenic gene-expression approach using microinjection of a gene of

interest into eggs in conjunction with a transposon _piggyBac_-based system15, the tremendous capabilities of silk production can now be applied to recombinant protein production. Using

these methods, _B. mori_-produced interferon (IFN) was introduced as the first antiviral drug for veterinary use, and two _B. mori_-produced IFNs are currently on the market16. Protein

production in _B. mori_ enables breeding at high density in a closed system, higher expression efficiency than that by bacterial expression, less production differences between batches, and

protein post-translational modifications similar to those in mammals. In addition, the use of a GAL4/UAS system in combination with a _piggyBac_ system has enabled spatiotemporal-specific

expression of transgenes of interest17, which also facilitates easy purification of the recombinant protein. Since the development of these techniques, various kinds of recombinant proteins

have been expressed using organ-specific protein production18,19. In the case of protein production in _B. mori_, the biological activities of the recombinant proteins should be reevaluated.

In _B. mori_, two expression systems, a baculovirus-based expression system using silkworm as an expression host (baculovirus/silkworm expression system) and a transposon-based transgenic

gene expression system (transgenic silkworm expression system), are widely used, and the proteins produced by these systems might have different properties. Both expression systems enable

high protein production, but their respective effects on the recombinant protein qualities, especially post-translational modifications such as protein degradation, have not been examined.

In general, the baculovirus/silkworm expression system yields much greater amounts of protein than the transgenic silkworm expression system, but this augmented production might provoke

endoplasmic reticulum (ER) stress and/or dysfunction via unfolded protein accumulation, which in turn could trigger ER-associated degradation and/or an unfolded protein response to restore

ER homeostasis20,21. Indeed, co-expression of molecular chaperones with a target gene of interest has been shown to facilitate and enhance recombinant protein production in _B. mori_22,23,

suggesting that the endogenous quality-control machinery of the ER in _B. mori_ is not sufficient for the production of correctly folded proteins. Furthermore, the introduction of a

baculovirus into the baculovirus/silkworm expression system itself could severely damage _B. mori_ and induce cell death. If any of these potential hazards were to arise, the

baculovirus/silkworm expression system could produce low quality, post-translationally unprocessed, immature or proteolytically-degraded proteins. In this study, to focus on the quality of

the recombinant proteins produced in _B. mori_ by the two systems, we expressed bovine interferon γ (IFN-γ) as a model protein in the baculovirus/silkworm expression system and transgenic

silkworm expression system. The IFN-γ proteins were then evaluated in terms of two qualities: _N_-glycosylation and protein cleavage. Major differences were observed between the IFN-γ

produced by the baculovirus/silkworm expression system and that produced from the transgenic silkworm expression system. This study introduces new concerns that must be considered when

producing biopharmaceutical proteins using _B. mori_ as an “insect factory”. RESULTS COMPARISON OF RECOMBINANT IFN-Γ PRODUCTION IN TWO PROTEIN-EXPRESSION SYSTEMS Two major protein-expression

systems utilizing _B. mori_, a baculovirus-based expression system using the silkworm as an expression host (baculovirus/silkworm expression system) and a transgenic gene expression system

employing microinjection of a gene of interest into eggs (transgenic silkworm expression system), used to express bovine IFN-γ as a model protein. IFN-γ is the primary macrophage-activating

cytokine and transduces signals essential for an innate immune response24. IFN-γ consists of 166 aa containing a 23 aa N-terminal signal peptide for secretion from activated T lymphocyte

cells and also the KRKR sequence for nuclear localization in the region proximal to the C-terminus25 (Fig. 1A). IFN-γ possesses two potential _N_-glycosylation sites, Asn39 and Asn106,

suggesting that IFN-γ is produced as an _N_-glycoprotein in _B. mori_. It should be noted that _O_-glycosylation of IFN-γ has not yet been identified. IFN-γ was successfully produced in both

systems and purified using cation exchange chromatography. The yields of purified IFN-γ produced by the baculovirus/silkworm expression system and the transgenic silkworm expression system

were 580 μg and 2.5 μg per larva, respectively. Thus, focusing only on the production, the baculovirus/silkworm expression system exhibited the superior production system by far.

Interestingly, both baculovirus/silkworm expression and transgenic silkworm expression systems produced several different forms of IFN-γ; the transgenic silkworm expression system produced

three forms of IFN-γ (tIFN-γ) (bands a to c in Fig. 1B), whereas the baculovirus/silkworm expression system produced four forms of IFN-γ (bIFN-γ) (bands A to D in Fig. 1B and Supplementary

Data File 1). IFN-γ has two _N_-glycosylation sites, meaning that three isoforms could be produced in theory: IFN-γ with _N_-glycosylation at both _N_-glycosylation sites, with

_N_-glycosylation at either _N_-glycosylation site, and without _N_-glycosylation. To confirm the _N_-glycosylations on IFN-γs, de-glycosylation analysis using peptide: _N_-glycosidase F

(PNGase F) and glycan staining were performed (Fig. 1C and Supplementary Data File 1). PNGase F digestion converged _N_-glycosylated tIFN-γs to completely deglycosylated forms which were

negative for _N_-glycan staining (Fig. 1C, right panel), whereas bIFN-g still had PNGase F-insesitive _N_-glycans (Fig. 1C, left panel). The molecular mass of the de-glycosylated form of

tIFN-γ agreed well with the calculated mass of mature IFN-γ without a signal peptide (16.8 kDa). Meanwhile, deglycosylated bIFN-γ still exhibited two bands, one of which showed the same

molecular mass as the de-glycosylated forms of tIFN-γ. In addition, the predominant de-glycosylated form of bIFN-γ was the band showing a faster electro-mobility shift, and the upper

de-glycosylated form gave only a faint signal. These results indicated that the transgenic silkworm expression system produced isoforms of IFN-γ with different _N_-glycosylation profiles.

However, based on the amino acid sequence of IFN-γ, the four forms detected in bIFN-γ were not caused by _N_-glycosylation itself, because IFN-γ had no other rare potential _N_-glycosylation

sites, N-X-C, N-X-V, and N-G26 and _C_-mannosylation site, W-X-X-W/C (X represents any amino acid)27. This evidence strongly suggested that bIFN-γ was not only _N_-glycosylated but further

post-translationally modified by presumably proteolytic degradation(s). STRUCTURAL DIFFERENCES OF _N_-GLYCAN ON RECOMBINANT IFN-Γ Purified IFN-γs from both systems showed not only different

molecular masses and numbers of bands but also bands with different features: bIFN-γ showed clear bands, whereas tIFN-γ showed broader bands (Fig. 1B). This suggested two possibilities:

either the band differences were attributable to the differences in the number of _N_-glycan structures or to the differences in the structures themselves. That is, either the number of

_N_-glycan structures on bIFN-γ was smaller, or the size of the _N_-glycan structures on tIFN-γ was larger, and/or the difference of _N_-glycan structures might have affected the band

patterns. In fact, focusing on the _N_-glycan difference, there was a difference of approximately 1 kDa in the molecular masses of the pauci-mannose-type structure(s) and high-mannose-type

structures, especially between Man2GlcNAc2 (Man, mannose; GlcNAc, _N_-acetylglucosamine) (M2) and M8/M9. To investigate the _N_-glycan effect(s) on the variance of IFN-γ, _N_-glycans from

each band were prepared and labeled with a fluorescence tag of 2-aminoprydine (PA), then analyzed by reverse phase (RP)-high performance liquid chromatography (HPLC) and liquid

chromatography-tandem mass spectrometry (LC–MS/MS) (Fig. 2 and Table 1). Focusing on each IFN-γ, the _N_-glycan structures on each band and their ratio were similar to each other, but they

were dissimilar between bIFN-γ and tIFN-γ. Most _N_-glycans on bIFN-γ were pauci-mannose-type structures, whereas the _N_-glycans on tIFN-γ were modified with terminal GlcNAc residue(s). The

highest molecular mass _N_-glycan structure was M5 on tIFN-γ, and the ratio was quite low, indicating that the presence of variants detected as different bands was derived not from the

difference of _N_-glycans but rather from _N_-glycosylation and/or other post-translational modification(s) of IFN-γs. The predominant _N_-glycans on bIFN-γ and tIFN-γ were M2B and GNM3B,

respectively. Notably, the _N_-glycans from bIFN-γ included no GlcNAc-terminal _N_-glycans. The total number of structures on bIFN-γ was much smaller than that of tIFN-γ. These results were

also supported by the fact that both the values of _N_-glycan dispersity on bIFN-γ calculated from the molecular weight and their ratio of _N_-glycans were smaller than those for tIFN-γ

(Table 1). Thus, _N_-glycan on bIFN-γ had less heterogenicity than that on tIFN-γ. SITE-SPECIFIC _N_-GLYCOSYLATION ANALYSIS OF IFN-Γ CBB staining demonstrated that bIFN-γ and tIFN-γ had

different _N_-glycosylation variants (Fig. 1b). To confirm the _N_-glycosylation status on Asn39 and Asn106, the bands were further analyzed by tryptic in-gel digestion, followed by

nanoLC-MS/MS analysis and MASCOT search (Fig. 3). No signal peptides were detected on bIFN-γ and tIFN-γ, indicating that bIFN-γ and tIFN-γ were secreted in _B. mori_ as in mammals. The

N-terminal peptide detected in a database search revealed that bIFN-γ and tIFN-γ were modified with the same machinery. Focusing on the bands A and a, neither of the peptides carrying

putative _N_-glycosylation sites was hit in the MASCOT search, indicating that Asn39 and Asn106 on bIFN-γ and tIFN-γ were fully _N_-glycosylated. This is because peptide hits in MASCOT

search indicates that the MS/MS results are consistent with the calculated molecular weight of the peptide without _N_-glycans. On the other hand, bands B, C, and b were hit in the MASCOT

search. De-glycosylation analysis exhibited that these bands were certainly _N_-glycosylated (Fig. 1C), suggesting that the difference of molecular mass of these bIFN-γ and tIFN-γ variants

was derived from heterogenicities of _N_-glycosylation; one of the two potential _N_-glycosylation sites was _N_-glycosylated. To elucidate _N_-glycosylation on each peptide in more detail,

_N_-glycopeptides were analyzed using datasets from nanoLC-MS/MS analysis (Fig. 4, and Supplemental Fig. S1 and Table S1). Asn39 on bIFN-γ in bands A-C had the M2F structure as the

predominant structure, whereas, although some of the _N_-glycan contaminants, possibly from band C, were detected, Asn39 on bIFN-γ in band D was hardly _N_-glycosylated. Through bands A-C,

the _N_-glycan structures and their ratio were similar. Focusing on non-_N_-glycosylated Asn39, bands B and C also had the peptide without _N_-glycosylation, _m/z_ 2053 from

EIENLKEYFN39ASSPDVAK, whose ratio increased from band B to D. In Asn106, the predominant structures on each band were M2. Although _N_-glycan structures and their ratio were almost identical

to those detected in Asn39, the ratio of non-_N_-glycosylated peptide drastically increased from band B to C, and finally only EIENLKEYFN39ASSPDVAK peptide was detected in band D. These

results indicated that the _N_-glycosylation variants of band C in bIFN-γ were mainly derived from _N_-glycosylation on Asn39 and non-_N_-glycosylation on Asn106. In tIFN-γ, band a was fully

_N_-glycosylated, and the predominant structure was GNM3 at both Asn39 and Asn106 (Fig. 4 right panel, and Supplemental Fig. S1 and Table S1). However, the _N_-glycan structures and the

ratios were slightly different. Asn39 was modified with GlcNAc-terminal and/or fucosylated _N_-glycans, whereas the ratio of GlcNAc-terminal _N_-glycans was decreased by 68% in comparison

with Asn39, and fucosylated _N_-glycans were not detected on Asn106. This result demonstrated that the fucosylated _N_-glycans, which were mostly α1,6-linked fucose (Fuc), detected in HPLC

analysis in Fig. 2 were derived from Asn39. Interestingly, not only non-_N_-glycosylated peptide but also the peptide retaining a single GlcNAc residue were detected on both

_N_-glycosylation sites in band b. Finally, only the non-_N_-glycosylated and predominant peptides with a single GlcNAc residue were detected in band c. The ratios of non-_N_-glycosylated

peptides were not significantly changed, but the ratio of the peptides retaining a single GlcNAc residue increased, indicating that the degrees of _N_-glycosylation on Asn39 and Asn106 were

approximately similar between bands b and c, but de-glycosylation resulted in the formation of band c from band b. Thus, the decisive difference between band a–c was derived from a further

truncation of _N_-glycans on both _N_-glycosylation sites rather than a difference in the _N_-glycosylation state. These results also provided the evidence that most potential

_N_-glycosylation sites on tIFN-γ were completely _N_-glycosylated once. It should be noted that this _N_-glycan truncation was not observed in band B–D in bIFN-γ. IDENTIFICATION OF CLEAVAGE

SITES IN IFN-Γ De-glycosylation analysis of IFN-γs by PNGase F digestion revealed two forms of bIFN-γ, suggesting that bIFN-γ had two different peptide backbones (Fig. 1C). The upper band

of de-glycosylated bIFN-γ exhibited the same electro-mobility shifts as de-glycosylated tIFN-γ. Peptide mapping analysis of trypsin-digested IFN-γs provided the evidence that the N-terminal

peptides were the same among bands A-D (Fig. 3). Remarkably, although the N-terminal peptides detected by peptide mapping were the same, the C-terminal peptide identified in bands a-c,

S155QNLFR160, was identified in band A but not in the peptides from bands B-D. This demonstrated that bIFN-γ consisted of two peptide forms: a mature and full length IFN-γ and a

C-terminal-truncated IFN-γ. Furthermore, the major de-glycosylated form of bIFN-γ showed the same electro-mobility shift as band D, indicating that the upper band of de-glycosylated bIFN-γ

was from band A and the lower band of de-glycosylated bIFN-γ was from bands B-D. The molecular masses of bands A and a were different, but the estimated difference of approximately 0.92 kDa

almost agreed with the difference of the calculated mass of _N_-glycan structures between bIFN-γ and tIFN-γ: 0.73 kDa on average. These results indicated that the peptide backbones of band A

in bIFN-γ and tIFN-γ were the same, and the difference in molecular mass between bands A and a was due to the difference of the _N_-glycan structure. Unfortunately, the cleavage site(s) of

the C-terminus in bIFN-γ was not determined in this analysis due to the lack of appropriate peptidases for in-gel digestion, but it could be concluded that most of the bIFN-γs were

proteolytically-cleaved somewhere in S147NLRKRKRSQNLFR160. The de-glycosylation product of tIFN-γ also showed two close bands, namely band X and band Y with a ratio of 0.65:0.35 (Fig. 5A).

To confirm the cleavage site, both bands were excised, followed by in-gel digestion by Lys-C, nanoLC-MS/MS analysis, and an annotation by peptide mapping (Fig. 5B). Lys-C revealed that the

N-terminal sequence of both bands was the same, G25QFFREIENLK35, suggesting that the C-terminal region of tIFN-γ was cleaved in two different positions. A more detailed search for MS

analysis in band Y led to the identification of a peptide corresponding to the mass of SQNL(/I)FRG, _m/z_ 976.5. The fragmentation of the peptide by MS/MS demonstrated that the peptide

certainly consisted of SQNL(/I)FRG (Fig. 5C). This peptide hit the sequence from Ser154 to Gly161 of IFN-γ. Focusing on the band X, a longer peptide corresponding to the mass of

SQNL(/I)FRGRRA, _m/z_ 1359.7, was identified and its sequence was also identical (Supplementary Fig. S2). These results indicated that tIFN-γ was cleaved at two different C-terminal sites,

Gly161 and Ala164. DISCUSSION Following the first report of IFN-β expression in Sf21 cells using baculovirus in the early 1980s28, insects have been widely used as “factories” to produce

recombinant proteins6. Starting with the first recombinant protein of IFN-α produced using _B. mori_ nucleopolyhedrovirus in 198413,14 and the establishment of methods for the generation of

transgenic silkworms in 200015, _B. mori_ also became a candidate for a promising platform for recombinant protein production. Over the last three decades, therefore, _B. mori_ has been used

for the production of various kinds of recombinant proteins as model proteins, including fluorescent proteins, immunoglobulins, growth factors and cytokines essential for mammalian

proliferation16,18,19. In addition, the _B. mori_ baculovirus/silkworm expression system produces a target protein and/or peptide within a few days, enabling quick responses to seasonal

diseases and epidemics. In fact, the SARS-CoV-2 spike protein has already been produced for research of and/or application to COVID-19 vaccines29. Currently, many companies are actively

trying to produce recombinant proteins in larvae using baculovirus/silkworm expression systems30. Among the commercially available proteins produced in _B. mori_ larvae are the IFNs produced

by Toray Industries (Tokyo) for veterinary use30. In addition to the various efforts to realize rapid and large-scale recombinant protein production by insect baculovirus/silkworm

expression systems, _N_-glycosylation of recombinant proteins has also been taken into consideration. _N_-Glycosylation of proteins determines in vivo activities and stabilities, and thus it

is imperative insect-type _N_-glycans should be optimized to mammalian type. In the transgenic silkworm expression system, it has become possible to produce a protein-of-interest in each

organ using an organ-specific promoter, such as the promoters of _ser1_ and _fibH_, thereby controlling the _N_-glycan structure to some extent31,32. However, to our knowledge there has been

no comprehensive analysis to compare the qualities and quantities of identical proteins expressed in the baculovirus/silkworm expression system and transgenic silkworm expression system. To

facilitate the use of the recombinant proteins produced in these two _B. mori_ expression systems, more detailed analyses, especially with respect to the protein qualities, will be needed,

along with a thorough reevaluation of these two expression systems. Here, focusing on the characteristics of the recombinant proteins, we expressed IFN-γ using two different expression

methods in _B. mori_. Both expression systems successfully produced IFN-γ, but the number of variants and their molecular weights were dissimilar. The putative structures of the IFN-γs with

_N_-glycans expressed in this study are shown in Fig. 6. A previous study also demonstrated that an insect-produced IFN-γ analyzed using a baculovirus expression vector system showed several

bands representing the different levels of _N_-glycosylation33. This heterogeneous _N_-glycosylation was due to underglycosylation, and it has also been observed when IFN-γ was expressed in

leukocytes34, CHO cells35,36,37,38, and plants39. Detailed _N_-glycosylation and _N_-glycan analyses of both bIFN-γ and tIFN-γ revealed that potential _N_-glycosylation sites were

_N_-glycosylated in the same manner, but the structures were different; bIFN-γ had pauci-mannose type structures of M2B or M2FB and the number of structures was small, whereas tIFN-γ had

GlcNAc-extended structures (Table 1). This was one of the reasons that variants with different molecular weights were produced. The results of _N_-glycan staining also suggested that the

number of _N_-glycan structures was smaller on bIFN-γ, since the bands for bIFN-γ were narrower than those for tIFN-γ (Fig. 1C). A previous report demonstrated that INF-γ expressed in Sf9

cells possessed M3 and M3F as predominant structures on Asn39 and Asn 106, respectively36. _N_-Glycans on IFN-ω produced in insect cells, as another example, contained M2F and M3F

structures, while natural IFN-ω carried complex-type _N_-glycans40. Thus, this pauci-mannosidic structure on recombinant _N_-glycoprotein is due to the baculovirus/silkworm expression

system. _N_-Glycan of native human IFN-γ produced in CD8+ T lymphocytes showed more than 30 _N_-glycan structures, among which some of the _N_-glycan was sialylated with _N_-acetylneuraminic

acid34. Thus, it is considered to be essential to produce sialylated IFN-γ for in vivo activity. Indeed, IFN-γ was also produced in _E. coli_, but the half-life of _E. coli_-produced IFN-γ

was short, presumably due to the underglycosylation caused by lack of _N_-glycosylation41. Recombinant IFN-γ with oligo-mannosidic _N_-glycan produced in insect cells had a negative effect

on the stability of the IFN-γ in bloodstream circulation, and the IFN-γ was eliminated more rapidly than native IFN-γ42. From the point of view of _N_-glycosylation and _N_-glycan, target

protein production by a transgenic silkworm expression system enabling organ-specific protein production is preferable. The transgenic silkworm expression system especially in middle silk

gland (MSG) resulted in the accumulation of _N_-glycans with terminal GlcNAc residue(s), which is indispensable for biosynthesis of mammalian-type _N_-glycan31. Indeed, tIFN-γ expressed in

MSG using the GAL4/UAS system and _ser1_ promoter had larger amount of _N_-glycans with terminal GlcNAc residues (Table 1 and Supplementary Table S1). Taking into consideration the further

_N_-glycosylation and the structural modification of _N_-glycan, it could be concluded that a transgenic silkworm expression system which can produce a protein with an _N_-glycan structure

closer to that of the mammalian type is more suitable for protein production compared to a baculovirus/silkworm expression system with a high production level. Interestingly, a single GlcNAc

residue on an _N_-glycosylation site was detected in both Asn39 and Asn106 of tIFN-γ. This unusual GlcNAc modification has also been reported in some plant proteins43,44. As mentioned

above, heterologously expressed IFN-γs had _N_-glycosylation variants due to the underglycosylation34,35,37,38,39, but this single GlcNAc residue on _N_-glycosylation sites and the presence

of IFN-γ with different molecular masses due to this single GlcNAc modification has not been reported so far. In general, sequential reactions of glycosyltransferases of ALG family proteins

in the ER catalyze the biosynthesis of Glc3Man9GlcNAc2 on dolichol (Dol)45 and the intermediates of this oligosaccharide are hardly detected except for the _alg_ mutant.

Glc3Man9GlcNAc2-PP-Dol, the most optimal substrate of the oligosaccharyltransferase (OST), is transferred to nascent polypeptides by the action of OST complex. In this oligosaccharide

transfer, the terminal α1,2-linked glucose is necessary for efficient _N_-glycosylation mediated by OST46. GlcNAc2-PP-Dol is also used as a minimal donor substrate, however, GlcNAc-PP-Dol is

poorly utilized by OST compared to GlcNAc2-PP-Dol although this GlcNAc residue is important for the catalytic function of OST47. These facts suggest that the GlcNAc residue on tIFN-γ is not

synthesized by incomplete _N_-glycosylation using GlcNAc-PP-Dol as a donor, but is a product that is decomposed after sugar chain synthesis. Therefore, _B. mori_ might have

endoglycosidase(s) contributing to the hydrolysis of the glycosidic bond between R-GlcNAcβ1,4-GlcNAc-Asn. One candidate for the endoglycosidase is _endo_-β-_N_-acetylglucosaminidase

(ENGase). ENGase is categorized into two glycoside hydrolase (GH) families, GH18 and GH85, in the carbohydrate-active enzymes (CAZy) database (http://www.cazy.org). GH85 is widely

distributed from bacteria, through fungi, to eucaryotes48. Insects also have ENGase, and a putative _B. mori_ ENGase (Gene Model ID KWMTBOMO10033) was also identified as a GH85 protein in a

database search using KAIKObase (https://kaikobase.dna.affrc.go.jp/). Thus, _N_-glycans on tIFN-γ might be cleaved off by the action of this putative _B. mori_ ENGase. It has hypothesized

that this ENGase contribution to the production of a single GlcNAc modification also occurs in plants43,44. This uncommon modification of the GlcNAc residue on _N_-glycosylation site(s)

would also affect the stability of tIFN-γ. However, the GlcNAc residue also could be applied to _N_-glycoprotein remodeling by chemoenzymatic transglycosylation. Transglycosylation mediated

by ENGase activities with highly active intermediates, _e.g._, glycan oxazolines, has been applied to the chemoenzymatic synthesis of _N_-glycoproteins with a structure consisting of

homogeneous _N_-glycoforms49,50. Therefore, modification of the GlcNAc residue on tIFN-γ with sialylated _N_-glycans by transglycosylation would enable the production of more reliable IFN-γ

with improved stability in bloodstream circulation. The most significant and critical difference in the IFN-γs produced by the two different systems concerned the cleavage and/or degradation

of the C-terminal region (Fig. 5). IFN-γ has two important peptides: a secretion signal at the N-terminus and a propeptide at the C-terminus41. The C-terminal sequence of tIFN-γ,

S154QNLFRG161, agreed well with the native IFN-γ, suggesting that _B. mori_ has the same IFN-γ maturation machinery as mammals. Therefore, from the protein-maturation point of view, the

transgenic silkworm expression system using genetic transformation is preferable to the baculovirus/silkworm expression system. On the other hand, S154QNLFRGRRA164 was also detected,

suggesting tIFN-γ might be maturated via two different machineries in _B. mori_, although if so, the details are not yet clear. This was also suggested by the results for bIFN-γ, because

bIFN-γ was produced as a more truncated, proteolytically-cleaved or degraded form than tIFN-γ. The C-terminal effect of INFγ on the activity has been controversial. In fact, some

C-terminal-truncated variants, which are presumably the same as those observed in tIFNγ, have been reported in natural IFN-γ51,52, and this unexpected C-terminal nine-amino-acid truncation

was also observed in insect-produced IFN-ω40. However, a previous report demonstrated that truncation of the C-terminal flexible domain consisting mainly of Lys and Arg in IFN-γ contributes

to the stability of the molecule, and thereby enhances the solubility of IFN-γ53, but this truncation of the C-terminal flexible domain also induces a substantial abolishment of biological

activity, a phenomenon which becomes significant when there are twelve or more amino-acid deletions from the C-terminus53. Furthermore, bIFN-γ presumably lacked K150RKR153, which functions

as a nuclear localization signal and is essential for the nuclear translocation and cytokine function of IFN-γ25,54. These facts suggested that bIFN-γ is less functional and/or has no in

vivo activity due to the loss of the C-terminus. It has not been clarified whether this C-terminal truncation occurs only in IFN or also takes place in other proteins when the target protein

is expressed using a baculovirus expression system. As for the truncation of several amino acid residues, the higher the molecular mass of the produced protein, the more difficult it is to

evaluate the difference from that of the native protein, which results in several amino acid truncations being overlooked. Although this phenomenon might be limited to IFN-γ, and other model

proteins expressed in _B. mori_ should be evaluated, the truncation of amino acids in the baculovirus expression system might need to be taken into consideration, along with

post-translational modifications in protein production. METHODS MATERIALS The YMC*GEL Silica 12 nm S-50 μm column was purchased from YMC (Kyoto, Japan). Chelating Sepharose FF, the HiPrep

26/10 desalting column (Cytiva, Tokyo, Japan), HiPrep 16/10 S FF, and HiLoad 16/10 SP Sepharose High Performance were purchased from Cytiva. 2-Aminoprydine (PA) was purchased from FUJIFILM

Wako Chemicals (Osaka, Japan). PA-labeled sugar chains were purchased from TaKaRa Bio (Shiga, Japan) and Masuda Chemical Industry (Kagawa, Japan). EXPRESSION, PURIFICATION, AND

QUANTIFICATION OF IFN-Γ For the production and purification of bIFN-γ, the recombinant virus of cathepsin-deficient strain was injected into 50 silkworm larvae at the second day of fifth

instar (5.0 × 104 pfu/larva). Six days after injection, hemolymph was collected with the addition of a small amount of phenylthiourea (FUJIFILM Wako). Then, the harvested hemolymph was

clarified by low-speed centrifugation and subsequent ultracentrifugation. The supernatant of hemolymph was diluted 1:4 with 50 mM Tris–HCl pH 8.0 and applied on the YMC*GEL Silica 12 nm S-50

 μm column, which was equilibrated with 50 mM Tris–HCl pH 8.0. After washing the column with 50 mM Tris–HCl pH 8.0 containing 1 M NaCl, bIFN-γ was eluted with 50 mM Tris–HCl pH 8.0

containing 3 M NaCl and 30% ethylene glycol. The bIFN-γ fractions were diluted 1:9 with 50 mM phosphate buffer pH 7.0 and applied on the Chelating Sepharose FF column with CuSO4-5H2O, which

was equilibrated with 50 mM phosphate buffer pH7.0. The purified bIFN-γ was recovered with 50 mM phosphate buffer pH7.0 and concentrated with VIVASPIN 20 MWC 10,000 (Cytiva). Concentrated

bIFN-γ was desalted to DW with a HiPrep 26/10 desalting column. The amount of protein was quantified by using a BCA Protein Assay kit (Thermo Fisher Scientific, Tokyo, Japan). For the

transgenic silkworm expression, the bovine IFN-γ gene was amplified from the ORFeome Collaboration clone, ORH24802P (Promega, Madison, WI). The _Bln_I fragment of this PCR product was

inserted into the _Bln_I site of pBac[SerUAS/3xP3EGFP]55 to generate pBac[SerUAS_IFN-γ/3xP3-EYFP]. Transgenic silkworms were generated as previously reported using the plasmid

pBac[SerUAS_IFN-γ/3xP3-EYFP] as a vector15. Then the obtained transgenic silkworm lines harboring the IFN-γ gene under the regulation of a UAS sequence were mated with adults from the

Ser1-GAL4 strain, which carried a GAL4 gene driven by the sericin1 promoter. The middle glands isolated from transgenic silkworms were immersed in 20 mM phosphate-buffer, pH 7.2, and gently

shaken for 90 min in an ice bath. The resulting extract was centrifuged at 10,000×_g_ for 30 min and the supernatant fraction containing the extracted proteins was collected. The extraction

solution was frozen overnight, and the high-molecular-weight fiber proteins precipitated after thawing were removed by centrifugation. The extracted solution was coarsely fractionated by

ammonium sulfate at a concentration of 60%-80%. The precipitated crude fraction components were subjected to intermediate purification by cation chromatography using a cation exchange column

(HiPrep 16/10 S FF). Fractions with high expression product content were subjected to final purification using a cation exchange column (HiLoad 16/10 SP Sepharose High Performance).

_N_-GLYCOSYLATION ANALYSIS OF IFN-Γ _N_-Glycans on IFN-γs were digested using Peptide:_N_-glycosidase F (TaKaRa Bio, Shiga, Japan). _N_-Glycosylated and de-glycosylated IFN-γs were desalted

and concentrated by acetone precipitation, followed by SDS-PAGE separation using a SuperSep™ Ace 15% Gel (FUJIFILM Wako Chemicals, Osaka, Japan), CBB R-250 staining or _N_-glycan staining

using G.P.Sensor (J-OIL MILLS, Tokyo, Japan) and a POD immunostain kit (FUJIFILM Wako Chemicals). Digital images of CBB staining gels and stained blots were obtained using scanner software

and processed via Adobe Photoshop v. 22.5.0. The ratio of the two PNGase F-digested tIFN-γ s was determined from their band intensities using Image J56. _N_-GLYCAN ANALYSIS OF IFN-Γ IFN-γs

were separated by SDS-PAGE using a SuperSep™ Ace 15% Gel and stained with CBB R-250. The CBB-stained bands corresponding to the IFN-γs were excised from the gel and cut into small pieces,

then de-stained completely with 50 mM NH4HCO3 in 50% acetonitrile and dehydrated twice using acetonitrile. The proteins in the gels were in-gel digested using Trypsin Gold (Promega) in

ProteaseMAX™ Surfactant (Promega) at 50 °C for 1 h. The supernatants and extracts of gel pieces dehydrated using acetonitrile were lyophilized overnight. The resulting material was subjected

to hydrazinolysis, followed by lyophilization, _N_-acetylation, desalting with Dowex 50 × 2 (Muromachi Kagaku Kogyo, Kyoto, Japan), PA-labeling, and purification57. The PA-sugar chains were

detected by RP-HPLC using a HITACHI LaChrom HPLC system equipped with fluorescence58. Briefly, the mobile phase was composed of 0.02% trifluoroacetic acid (solvent A) and acetonitrile/0.02%

trifluoroacetic acid (solvent B) (20/80, v/v). RP-HPLC was performed using a Cosmosil 5C18-AR-II column (4.6 × 250 mm; Nacalai Tesque, Kyoto, Japan) with a HITACHI LaChrom HPLC system by

linearly increasing the solvent B concentration from 0 to 25% over 25 min at a flow rate of 0.7 ml/min. The eluted fractions were monitored by measuring the fluorescence intensity using

excitation and emission wavelengths of 310 and 380 nm, respectively. LC–MS/MS ANALYSIS AND STRUCTURAL DETERMINATION OF PA-SUGAR CHAINS The structural determination of PA-sugar chains chain

was performed as previously reported58. Briefly, LC–MS/MS was performed using an Agilent Technologies 1200 series instrument (Agilent Technologies, Santa Clara, CA) equipped with HCT plus

software (Bruker Daltonics, Bremen, Germany). For the LC, the mobile phase was composed of acetonitrile/acetic acid (solvent A: 98/2, v/v) and water/acetic acid/triethylamine (solvent B:

92/5/3, v/v/v). A Shodex Asahipak NH2P-50 2D column (2.0 mm ID × 150 mm; SHOWA DENKO) was used as an analytical column. The concentration of solvent B was increased in a linear gradient the

concentration from 20 to 55% over 35 min at a flow rate of 0.2 mL/min. The MS/MS analysis was performed in the positive-ion mode using the following parameters: scan range _m/z_ 350–2750;

nebulizer flow of 5.0 psi; dry gas flow rate of 3.0 L/min; dry temperature of 300 °C; target count of 200,000; and MS/MS Frag. Ampl. of 1.0 V. The relative amount of _N_-glycan was

calculated on the basis of the peak area of the LC. Based on the possible structure deduced from LC–MS/MS analysis, each structure of PA-sugar chains separated and collected using normal

phase-HPLC58 was compared with that of authentic PA-sugar chains. Non-commercially available PA-sugar chains were prepared as previously reported using glycosidases, such as mannosidase and

_N_-acetyl-hexosaminidase, and commercially available or prepared PA-sugar chains from _B. mori_ 58. PEPTIDE AND _N_-GLYCOPEPTIDE ANALYSIS OF IFN-Γ _N_-Glycopeptide analysis was performed in

the same manner as previously reported58. Briefly, small gel pieces of IFN-γ were prepared as described above. The proteins in the gels were in-gel digested using Trypsin Gold (Promega) in

ProteaseMAX™ Surfactant (Promega) at 50 °C for 1 h or Lys-C (Promega) in ProteaseMAX™ Surfactant at 37 °C for 20 h. The supernatant was collected, and the reactions were terminated by the

addition of trifluoroacetic acid to a final concentration of 0.5%. The trypsinized or Lys-C digested products were analyzed using an Agilent Technologies 1200 series nanoLC system (Agilent

Technologies) equipped with a micrOTOF-QII TOF–MS (Bruker Daltonics). For the liquid chromatography portion of the analysis, ZORBAX 300SB-C18 (5 μm, 0.3 mm × 5 mm) and ZORBAX 300SBC18 (3.5 

mm, 75 μm × 150 mm) (Agilent Technologies) columns were used as the trapping and analytical column, respectively. The mobile phase was composed of 0.1% formic acid (solvent A) and

acetonitrile containing 0.1% formic acid (solvent B) for nanoLC and 0.1% trifluoroacetic acid (solvent C) for peptide trapping on the column. Following injection, the flow was directed to

the trapping column in solvent C at a flow rate of 10 μL/min. The peptides were separated by linearly increasing the solvent B concentration from 8 to 30% over 30 min at a flow rate of 600

nL/min, followed by washing with 95% solvent B for 5 min, and equilibration for 18 min at the initial flow. In the MS portion of the analysis, the MS/MS parameters were as follows: scan

range _m/z_ 50–4500; nebulizer flow of 1.0 bar; dry gas flow rate of 5.0 L/min; and dry temperature of 180 °C in the positive-ion mode. The MS data were analyzed using Data Analysis 4.0

software, BioTools, and SequenceEditor (Bruker Daltonics). _N_-GLYCOPROTEIN MODELING The _N_-glycoprotein models of IFN-γs with _N_-glycans were constructed using bovine IFN-γ (PDB id: 1D9C)

as a protein template and the GLYCAM server (http://glycam.org/). Figures were prepared using the PyMOL molecular graphics system, version 1.7.1.1. (http://www.pymol.org/). DATA

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ACKNOWLEDGEMENTS This study was supported by a grant from the Agri-Genome Project of the Ministry of Agriculture, Forestry and Fisheries of Japan and by the Scientific Technique Research

Promotion Program for Agriculture, Forestry, Fisheries and Food Industry. AUTHOR INFORMATION Author notes * These authors contributed equally: Hiroyuki Kajiura, Ken-ichiro Tatematsu and

Tsuyoshi Nomura. AUTHORS AND AFFILIATIONS * International Center for Biotechnology, Osaka University, 2-1 Yamada-Oka, Suita-Shi, Osaka, 565-0871, Japan Hiroyuki Kajiura & Kazuhito

Fujiyama * Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, 2-1 Yamada-Oka, Suita-Shi, Osaka, 565-0871, Japan Hiroyuki Kajiura & Kazuhito Fujiyama

* Division of Silk-Producing Insect Biotechnology, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki, 305-8634, Japan

Ken-ichiro Tatematsu & Hideki Sezutsu * Sysmex Corporation, 1548 Ooaza Shimookudomi, Sayama, Saitama, 350-1332, Japan Tsuyoshi Nomura & Akihiro Usami * Division of Biomaterial

Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki, 305-8634, Japan Mitsuhiro Miyazawa * Silk Science and

Technology Research Institute, 1053, Iikura, Ami-Machi, Ibaraki, 300-0324, Japan Toshiki Tamura * Osaka University Cooperative Research Station in Southeast Asia (OU:CRS), Faculty of

Science, Mahidol University, Bangkok, Thailand Kazuhito Fujiyama Authors * Hiroyuki Kajiura View author publications You can also search for this author inPubMed Google Scholar * Ken-ichiro

Tatematsu View author publications You can also search for this author inPubMed Google Scholar * Tsuyoshi Nomura View author publications You can also search for this author inPubMed Google

Scholar * Mitsuhiro Miyazawa View author publications You can also search for this author inPubMed Google Scholar * Akihiro Usami View author publications You can also search for this author

inPubMed Google Scholar * Toshiki Tamura View author publications You can also search for this author inPubMed Google Scholar * Hideki Sezutsu View author publications You can also search

for this author inPubMed Google Scholar * Kazuhito Fujiyama View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS H.K. and K.F. designed the

research with assistance from A.U., T.T., and H.S. K.T. and T.N. produced the recombinant proteins. H.K., T.N., and M.M. prepared the purified protein. H.K. performed all the other

experiments. H.K. and K.F. wrote the manuscript. All authors reviewed the manuscript. CORRESPONDING AUTHOR Correspondence to Kazuhito Fujiyama. ETHICS DECLARATIONS COMPETING INTERESTS The

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recombinant proteins produced by two different _Bombyx mori_ expression systems. _Sci Rep_ 12, 18502 (2022). https://doi.org/10.1038/s41598-022-22565-7 Download citation * Received: 04 June

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