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Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response - PubMed

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

Byung-Kwon Choi et al. Glycoconj J. 2008 Aug.

Abstract

Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.

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Figures

Fig. 1
Fig. 1

DNA sequence encoding the mature human lactoferrin

Fig. 2
Fig. 2

SDS-PAGE with Coomassie blue stain (a) and Western blot with antibodies αhLF (b) and αHCP (c) of samples at different purification steps. Lane 1 broad range molecular weight standards, lane 2 rhLF fermentation supernatant, lane 3 SP Sepharose Fast Flow eluant, lane 4 Heparin Sepharose 6 Fast Flow eluant. αHCP antibody against Pichia host cell proteins

Fig. 3
Fig. 3

MALDI-TOF spectra of non-sialylated (a) and sialylated (b) rhLFs. Protein samples were diluted 1:1 with sinapinic acid in 0.1% trifluoroacetic acid and 50% acetonitrile, then spotted on a MALDI plate and examined using positive mode on an Applied Biosystems Bioanalyzer MALDI-TOF instrument

Fig. 4
Fig. 4

MALDI spectra of released glycan structures. Glycans were removed from rhLF with PNGase F and subjected to MALDI-TOF analysis in the positive ion mode for non-sialylated rhLF (a) and the negative ion mode for sialylated rhLF (b). The major peak observed in the non-sialylated rhLF N-glycan spectrum corresponds to Gal2GlcNAc2Man3GlcNAc2 (GS5.0), whereas the major peak in the sialylated rhLF N-glycan spectrum corresponds to Sia2Gal2GlcNAc2Man3GlcNAc2 (GS6.0). Red circles GlcNac, grey squares Man, violet squares Man, diagonal-striped squares Man, green diamond Gal, green four-pointed star Sia

Fig. 5
Fig. 5

Peptide mapping of non-sialylated rhLF. The dotted chromatogram displays the chromatographic UV trace for tryptic peptides from glycosylated rhLF; the solid line corresponds to the trace for deglycosylated tryptic peptides. Fractions from the glycosylated sample were then treated with PNGase F and run on liquid chromatography-coupled mass spectrometer via nanospray to identify peptide sequences. Fragments corresponding to deglycosylated and non-glycosylated N-glycosylation sites (N138, N479, N624) were identified in both samples and peaks containing these fractions were indicated with asterisks. Glycopeptides (N138 and N479) were identified in peaks marked with arrows

Fig. 6
Fig. 6

Reconstituting effect of lactoferrin on the secondary humoral immune response in mice suppressed by methotrexate. Splenocytes isolated from sheep red blood cell (SRBC) primed mice were incubated with SRBC, alone in the presence of methotrexate (MTX). Antibody forming cells (AFC) were evaluated after 4 days. Cells were cultured in the presence of non-sialylated LF (LF A), sialylated LF (LF B), or milk-derived LF (HLF Sigma). All LF concentrations were 1 μg/ml. The results are shown as mean values of AFC number from five wells±SE, calculated per 106 viable cells. Statistical analysis of groups: control vs. LF A NS; control vs. LF B NS; control vs. HLF Sigma p=0.0068; control vs. MTX p=0.0001; control vs. LF A+MTX p=0.0001; control vs. LF B+MTX NS; control vs. HLF Sigma+MTX NS; MTX vs. LF A+MTX NS; MTX vs. LF B+MTX p=0.0001; MTX vs. HLF Sigma+MTX p=0.0001 (ANOVA)

Fig. 7
Fig. 7

The activity of human recombinant lactoferrin in the secondary immune response in vitro is abolished by addition of sialic acid. Splenocytes isolated from sheep red blood cell (SRBC) primed mice were incubated with SRBC, alone in the presence of methotrexate (MTX). Antibody forming cells (AFC) were evaluated after 4 days. Cells were cultured in the presence of sialylated LF (LF B). Free sialic acid was added alone, or in combination with LF (a). Alternatively, anti-sialoadhesin monoclonal antibody CD169 (sialoadhesin) was added before LF (1:250) (b). a Control vs. LF B p=0.0001; control vs MTX p=0.0001; control vs. Sia NS; control vs. LF B+Sia NS; control vs. LF B+MTX NS; control vs LF B+Sia+MTX p=0.0001; MTX vs. LF B+MTX p=0.0001; MTX vs LF B+Sia+MTX NS; LF B+MTX vs. LF B+Sia+MTX p=0.0023 (ANOVA). b Control vs.LF B p=0.0291; control vs. MTX p=0.0001; control vs. Ab NS; control vs. LF B+Ab+MTX p=0.0001; LF B vs. LF B+Ab p=0.0001; MTX vs. LF B+MTX p=0.0001; MTX vs. LF B+Ab+MTX NS; LF B+MTX vs. LF B+Ab+MTX p=0.0001 (ANOVA)

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