Characterization of recombinant human lactoferrin N-glycans expressed in the milk of transgenic cows

Abstract

Lactoferrin (LF) is one of the most abundant bioactive glycoproteins in human milk. Glycans attached through N-glycosidic bonds may contribute to Lactoferrin functional activities. In contrast, LF is present in trace amounts in bovine milk. Efforts to increase LF concentration in bovine milk led to alternative approaches using transgenic cows to express human lactoferrin (hLF). This study investigated and compared N-glycans in recombinant human lactoferrin (rhLF), bovine lactoferrin (bLF) and human lactoferrin by Nano-LC-Chip-Q-TOF Mass Spectrometry. The results revealed a high diversity of N-glycan structures, including fucosylated and sialylated complex glycans that may contribute additional bioactivities. rhLF, bLF and hLF had 23, 27 and 18 N-glycans respectively with 8 N-glycan in common overall. rhLF shared 16 N-glycan with bLF and 9 N-glycan with hLF while bLF shared 10 N-glycan with hLF. Based on the relative abundances of N-glycan types, rhLF and hLF appeared to contain mostly neutral complex/hybrid N-glycans (81% and 52% of the total respectively) whereas bLF was characterized by high mannose glycans (65%). Interestingly, the majority of hLF N-glycans were fucosylated (88%), whereas bLF and rhLF had only 9% and 20% fucosylation, respectively. Overall, this study suggests that rhLF N-glycans share more similarities to bLF than hLF.

The N-glycans of lactoferrin: more than just a sweet decoration

“…The oligosaccharides released from Asn479 were composed of the rhLf neutral structures, whereas those from Asn138 were more complex and were mostly neutral and biantannary in nature(Yu et al 2011). The publication by Le Parc's group combined and expanded the knowledge about glycosylation of rhLf produced in transgenic cows(Le Parc et al 2017). They released N-glycans from rhLf by treatment with PNGase F, separated them from the proteins, and measured them by q-TOF LC/MS.…”

“…Each Lf (bLf, hLf, and rhLf) had similar high-mannose structures but with completely different abundance (Table 2). Le Parc and colleagues identified 10 mutual N-glycans for hLf and bLf, 9 for hLf and rhLf and 16 for bLf and rhLf(Le Parc et al 2017). Based on the Nglycan composition and abundance, their results suggested more similarities between rhLf and bLf than between rhLf and hLf (Table 2) (LeParc et al 2017).To sum up the results of Le Parc, hLf has a lot of sialylated structures, no significant amounts of high-mannose structures, and a high abundance of fucosylated oligosaccharides (LeParc et al 2017).…”

“…Le Parc and colleagues identified 10 mutual N-glycans for hLf and bLf, 9 for hLf and rhLf and 16 for bLf and rhLf(Le Parc et al 2017). Based on the Nglycan composition and abundance, their results suggested more similarities between rhLf and bLf than between rhLf and hLf (Table 2) (LeParc et al 2017).To sum up the results of Le Parc, hLf has a lot of sialylated structures, no significant amounts of high-mannose structures, and a high abundance of fucosylated oligosaccharides (LeParc et al 2017). By contrast, bLf has significant lower amounts of sialylated structures, it also contains the sialic acid N-glycolylneuraminic acid (Neu5Gc) in addition to Neu5Ac and it has high amounts of high-mannose N-glycans and very low amounts of fucosylated oligosaccharides.…”

“…N-Glycan composition and abundance of rhLf produced in transgenic cows based on(Le Parc et al 2017).…”

Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

“…However, the resulting product differs from the human one because of the different glycosylation pattern. 23 Similarly, partially "humanized" antibodies 24 produced in cattle for various purposes still display Neu5Gc epitopes 25,26 that might be the target of an immune response by the host with clinically relevant side effects.…”