Unexpected mucin-type O-glycosylation and host-specific N-glycosylation of human recombinant interleukin-17A expressed in a human kidney cell line

https://doi.org/10.1016/j.pep.2012.09.013Get rights and content

Abstract

The T helper cell-derived cytokine interleukin-17A (IL-17A) is a variably glycosylated disulfide-linked homodimer of 34–38 kDa. Its polypeptide monomer contains one canonical N-glycosylation site at Asn68, and human recombinant IL-17A was partly N-glycosylated when expressed in human kidney (HEK293) cells as a fusion protein with a melittin signal sequence and an N-terminal hexahistidine tag. Orbitrap mass analyses of the tryptic N-glycopeptide 63–69 indicated that the N-glycosylation was of the GalNAc-terminated type characteristic of cultured kidney cells. The mass spectrum of IL-17A monomer also included peaks shifted by +948 Da from the respective masses of unglycosylated and N-glycosylated polypeptides. These were caused by unpredicted partial O-glycosylation of Thr26 with the mucin-like structure -GalNAc(-NeuNAc)-Gal-NeuNAc. Identical O-glycosylation occurred in commercially sourced recombinant IL-17A also expressed in HEK293 cells but with a different N-terminal sequence. Therefore, the kidney host cell line not only imposed its characteristic pattern of N-glycosylation on recombinant IL-17A but additionally created an O-glycosylation not known to be present in the T cell-derived cytokine. Mammalian host cell lines for recombinant protein expression generally impose their characteristic patterns of N-glycosylation on the product, but this work exemplifies how a host may also unpredictably O-glycosylate a protein that is probably not normally O-glycosylated.

Highlights

► Human recombinant interleukin-17A expressed in HEK293 cells was variably glycosylated. ► Mass spectrometry was used to characterize its glycosylation. ► At the single N-glycosylation site, the pattern of glycosylation was kidney-cell typical. ► The single O-glycosylation at Thr26 was unexpected, and was of the mucin type.

Introduction

Mammalian host cell lines used for protein expression impose their own specific patterns of N-glycosylation on recombinant proteins [1], [2], [3]. Detailed studies of the cell-specificity of N-glycan structures are well advanced [4], [5], [6], but less is known about the cell-specificity of O-glycosylation, and O-glycosylation is generally less predictable. This note describes unexpected O-glycosylation of a human cytokine expressed in kidney cells. The result was observed in two versions of the recombinant product, one expressed in our own laboratories and another from a commercial source.

The protein in question was human interleukin-17A (IL-17A1; UniProt accession Q16552), a homodimeric cytokine of 34–38 kDa secreted from T helper cells, and originally named cytotoxic T-lymphocyte-associated antigen 8 (CTLA8) [7], [8], [9]. Loss of a 23-residue signal peptide [10], [11] shortens the 155 amino-acid gene product to a 132-residue polypeptide containing six cysteinyl residues that form two intrachain and two interchain disulfides [12]. IL-17A is the prototype of a class of proinflammatory cytokines that are targets for therapeutic intervention in rheumatoid arthritis and other autoimmune diseases mediated by T helper 17 cells [13], [14], [15], [16]. Interleukin-17F (IL-17F: UniProt accession Q96PD4), which is 55% sequence-identical to IL-17A, exists both as a homodimer and as a heterodimer with IL-17A. Much remains to be learned about the IL-17 family of cytokines and their corresponding receptors [17].

Sequence numbers in this note refer to full-length gene products including their signal peptides; we denote the N-terminal residue of IL-17A as Gly24 and the single canonical site for N-glycosylation as Asn68. For recombinant proteins with affinity tags at the N-terminus, sequence numbers for the mature protein match the natural sequence and tag-derived regions are described as N-terminal appendages.

The earliest reports of IL-17A expression in eukaryotic cell lines showed both that it was secreted as a mixture of glycosylated and unglycosylated disulfide-linked homodimeric proteins [8], [11], and that this pattern replicated the properties of the T cell-derived “natural” protein [11]. Authors have scrupulously avoided stating that N-glycosylation at Asn68 accounts for all of this heterogeneity, but this appears to be the prevalent view. O-Glycosylation has never been reported in IL-17A, and we failed to detect it in nonrecombinant protein immunoprecipitated from the conditioned medium of cultured T cells.

Peptide N-glycosidase F converted the upper band of an electrophoretic doublet of IL-17A expressed in Pichia pastoris to the lower band [18], but O-glycosylation was excluded based on the absence of any effect when the protein was treated with N-acetyl neuraminidase and an O-glycosidase. This conclusion has become questionable because the enzymes used would not be expected to remove the O-mannosyl glycans normally found in P. pastoris [19], [20]. By design, IL-17A made in P. pastoris had Ile20 as its N-terminal residue, with a yeast α-factor secretion signal removed from the recombinant gene product by proteolytic processing. Ile20 is also the N-terminal residue of a commercial preparation of human recombinant IL-17A produced in human 293 cells (Cell Signaling Technology, Danvers, MA).

While studying N-terminally His-tagged human recombinant IL-17A expressed in human kidney cells, we examined mass spectra of disulfide-reduced protein. Unglycosylated protein was detected with the correct mass, in addition to multiple species attributable to N-glycosylation of a type recently shown to be characteristic of kidney cells. Initially unexplained was a peak of +948 Da additional mass relative to unglycosylated peptide, and the same modification was also carried by a fraction of the N-glycosylated material. It was ultimately shown that the recombinant protein was partly O-glycosylated at Thr26, a previously unknown modification of this important research reagent.

Section snippets

Expression and purification of human recombinant IL-17A

Sequential overlapping PCR amplifications yielded a gene encoding a fusion protein composed of a honeybee melittin signal sequence (residues 1–21 of UniProt accession P01501) followed by a Gly-Ser-Gly spacer, a hexahistidine tag and a further Gly-Ser-Gly spacer, all followed by residues 20–155 of IL-17A (UniProt accession Q16552). This was cloned into the Sal I and Eco RI sites of a vector [21] containing a murine cytomegalovirus promoter followed by a tripartite leader sequence and a hybrid

Results

Earlier reports showed that Gly24 was the N-terminus of human recombinant IL-17A expressed with the natural biological signal peptide and purified after secretion from mouse NS0 cells [11] or Chinese hamster ovary cells [10]. In the present work, human recombinant IL-17A was expressed in HEK293 cells as a fusion protein equipped with a honeybee melittin secretion signal peptide and a hexahistidine tag placed N-terminal to the canonical sequence of IL-17A beginning at Ile20 (Fig. 1).

SDS–PAGE

Discussion

Since the earliest biochemical studies of IL-17A were performed, its variable glycosylation has been cautiously attributed to partial occupancy of the single canonical site for N-glycosylation at Asn68 [8], [11]. This made it a surprise to detect partial O-glycosylation at Thr26 in IL-17A secreted from HEK293 cells.

Recombinant IL-17A is a target of structural biology. Information about any microheterogeneity that it displays is pertinent to direct structural analyses by crystallography and

Acknowledgment

We thank Tom McLellan for assistance with mass spectrometry and related software.

References (32)

  • S. Wedepohl et al.

    N-Glycan analysis of recombinant L-selectin reveals sulfated GalNAc and GalNAc−GalNAc Motifs

    J. Proteome Res.

    (2010)
  • E. Rouvier et al.

    CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus Saimiri gene

    J. Immunol.

    (1993)
  • Z. Yao et al.

    Human IL-17: a novel cytokine derived from T cells

    J. Immunol.

    (1995)
  • P. Miossec

    Interleukin-17 in fashion, at last: ten years after its description, its cellular source has been identified

    Arthritis Rheum.

    (2007)
  • Z. Zhang et al.

    Signal peptide prediction based on analysis of experimentally verified cleavage sites

    Protein Sci.

    (2004)
  • F. Fossiez et al.

    T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines

    J. Exp. Med.

    (1996)
  • Cited by (12)

    • Conformational characterization of nerve growth factor- reveals that its regulatory pro-part domain stabilizes three loop regions in its mature part

      2017, Journal of Biological Chemistry
      Citation Excerpt :

      It is important to note that the identified glycoforms of proNGF expressed in CHO cells are likely not identical to the exact glycoforms of proNGF found in human brain tissue (58–60). Especially O-glycosylation sites have been shown to differ between recombinant protein and naturally derived protein material (61), and the identified O-glycosylations sites in the pro-part of proNGF can only be interpreted as an indication of the presence of O-linked glycosylations in human proNGF. Interestingly, the identification of O-linked glycosylations sites close to one of the mutated furin cleavage sites, however, could be biologically relevant, as O-linked glycosylation have been shown to be protective toward pro-protein convertases such as furin (62, 63).

    • A continuous and direct assay to monitor leucine-rich repeat kinase 2 activity

      2014, Analytical Biochemistry
      Citation Excerpt :

      Product fractions were evaporated to a final volume of 20 μl each and analyzed by LC–MS. LC–MS was conducted using 8-μl injections of either the whole digest or the product of Fe–NTA affinity capture. LC–MS was carried out by capillary flow reversed-phase high-performance liquid chromatography (HPLC) and an LTQ Orbitrap XL mass spectrometer, as described previously [14]. Database searches to identify peptides were conducted using Mascot (Matrix Science) [15].

    • Human IL-34 and CSF-1 establish structurally similar extracellular assemblies with their common hematopoietic receptor

      2013, Structure
      Citation Excerpt :

      Such MW differs by 5 kDa from the theoretical MW of 26 kDa. As O-glycosylation in HEK293 cells was recently shown to be of the GalNAc-NeuNAc-Gal-NeuNAc type (Geoghegan et al., 2013), we can deduce that at least five O-linked glycans must be present in the C-terminal segment of hIL-34FL. Our findings confirm that the C terminus of hIL-34FL is heavily glycosylated via O-linked glycans, providing insights into the already observed differences between IL-34 and CSF-1 in terms of functionality and signaling activation patterns (Chihara et al., 2010).

    View all citing articles on Scopus
    View full text