Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.

To identify polymorphisms associated with variability of iron overload severity in HFE-associated hemochromatosis, we performed exome sequencing of DNA from 35 male HFE C282Y homozygotes with either markedly increased iron stores (n=22; cases) or with normal or mildly increased iron stores (n=13; controls). The 35 participants, residents of the U.S., Canada, and Australia, reported no or light alcohol consumption. Sequencing data included 82,068 single nucleotide variants, and 10,337 genes were tested for a difference between cases and controls. A variant in the GNPAT gene showed the most significant association with severe iron overload (p = 3×10−6, p=0.033 by the likelihood ratio test after correction for multiple comparisons). Sixteen of 22 participants with severe iron overload had GNPAT polymorphism p.D519G (rs11558492) (15 heterozygotes, one homozygote). No control participant had this polymorphism. To examine functional consequences of GNPAT deficiency, we performed siRNA-based knockdown of GNPAT in the human liver-derived cell line HepG2/C3A. This knockdown resulted in a >17-fold decrease in expression of the mRNA encoding the iron regulatory hormone hepcidin. Conclusion: GNPAT p.D519G is associated with a high-iron phenotype in HFE C282Y homozygotes and may participate in hepcidin regulation.

Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.

BMP-SMAD signalling plays a crucial role in numerous biological processes including embryonic development and iron homeostasis. Dysregulation of the iron-regulatory hormone hepcidin is associated with many clinical iron-related disorders. We hypothesised that molecules which mediate BMP-SMAD signalling play important roles in the regulation of iron homeostasis and variants in these proteins may be potential genetic modifiers of iron-related diseases. We examined the role of endofin, a SMAD anchor, and show that knockdown of endofin in liver cells inhibits basal and BMP-induced hepcidin expression along with other BMP-regulated genes, ID1 and SMAD7. We show for the first time, the in situ interaction of endofin with SMAD proteins and significantly reduced SMAD phosphorylation with endofin knockdown, suggesting that endofin modulates hepcidin through BMP-SMAD signalling. Characterisation of naturally occurring SNPs show that mutations in the conserved FYVE domain result in mislocalisation of endofin, potentially affecting downstream signalling and modulating hepcidin expression. In conclusion, we have identified a hitherto unrecognised link, endofin, between the BMP-SMAD signalling pathway, and the regulation of hepcidin expression and iron homeostasis. This study further defines the molecular network involved in iron regulation and provides potential targets for the treatment of iron-related disorders.

During mitosis, the Golgi undergoes two sequential fragmentation steps to break from ribbon to individual stacks, then from stacks to vesicles. While the mechanism that regulates the first step has been studied, it remains obscure how the second vesiculation step is regulated. It has been suggested that Cdk1-dependent phosphorylation of the cis-Golgi matrix protein GM130 regulates the second step. Here we have tested if phorphorylation of GM130 by Cdk1 is required for Golgi vesiculation and mitotic progression. Inhibition of Cdk1 activity caused a failure of Golgi vesiculation and defects in chromosome congression/segregation. Expression of non-phosphorylatable mutant of GM130 (GM130S25A) in cells depleted of endogenous GM130 caused no apparent defects in Golgi vesiculation and mitotic progression. Similarly, no apparent defects in Golgi vesiculation and mitotic progression were observed when GM130S25A was expressed in GM130-deficient CHO cells. Our observations suggest that while Cdk1 based phosphorylation is essential for mitotic Golgi vesiculation, mammalian cells could possess redundant, S25 phosphorylation of GM130 independent pathways that ensure Golgi vesiculation and mitotic progression.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Variability in the severity of iron overload among homozygotes for the HFE C282Y polymorphism is one of the major unsolved problems in our understanding of hereditary hemochromatosis (HH). We previously conducted exome sequencing of DNA from 35 HFE C282Y male homozygotes with either markedly increased iron stores (n=22; cases) or normal to mildly increased iron stores (n=13; controls) to identify rare and common causal variants associated with variability of disease expression in HH. The 35 participants, residents of the U.S., Canada, and Australia, reported little or no alcohol consumption. Criteria for HFE C282Y homozygotes with increased iron stores included serum ferritin >1000 µg/L at diagnosis and either (a) hepatic iron concentration >236 µmol/g dry weight (reference range 0-36 µmol/g) or (b) mobilized body iron >10 g by quantitative phlebotomy. Criteria for HFE C282Y homozygotes with normal or mildly elevated iron stores included (a) serum ferritin <300 µg/L or either (a) age ≥40 y with ≤2.5 g iron removed by phlebotomy to achieve serum ferritin <50 µg/L, or (b) age ≥50 y with ≤3.0 g iron removed by phlebotomy. After quality control filtering, sequencing data included 82,068 single nucleotide variants and 1,403 insertions/deletions (indels); 10,337 genes were tested for a difference between cases and controls. We identified the polymorphism GNPAT p.D519G (1556A>G; exon 11; chromosome 1q42; rs11558492) as the most significantly different variant between cases and controls (p=0.033 by the likelihood ratio test after correction for multiple comparisons). In a principal components analysis of ancestry, all 35 study participants were clustered closely together within a larger group of Europeans. Mean (SD) ages at presentation were 53 (11.5) y and 57 (10.0) y for cases and controls, respectively. Median serum ferritin was 2391 µg/L in cases and 302 μg/L in controls. The median transferrin saturation (96%) was greater in cases than controls (70%). Sixteen of 22 case participants had polymorphism GNPAT p.D519G (rs11558492) (15 heterozygotes, 1 homozygote); no control participant had this polymorphism. The homozygous case presented at age 26 y with severe iron overload but no cirrhosis. One GNPAT p.D519G heterozygote presented at age 36 y with severe iron overload and cirrhosis. GNPAT p.D519G is common among people of European descent (allele frequency 20.6%) and might interact with aberrant HFE to increase the risk of hepatic iron overload. More recently, we compared the allele frequencies of GNPAT p.D519G in the present 22 cases and 13 controls with that of 4300 European Americans in the NHLBI Exome Sequencing Project Exome Variant Server. The allele frequency in cases was greater than that of European Americans (38.6% vs. 20.6%, respectively; p = 0.0076). The allele frequency in controls was significantly lower (0% vs. 20.6%, p = 0.0054). Next, to determine whether other known mutations influenced iron phenotypes, the exome data were used to screen for mutations in HAMP, HJV, TFR2, FPN1, and TMPRSS6. One case participant was heterozygous for HJV p.G320V; he was among the six case participants who did not have GNPAT p.D519G. No other known or probable mutation that would possibly explain differences in expression between cases and controls was found. To examine functional consequences of GNPAT deficiency, we now have performed siRNA-based knockdown of GNPAT in the human liver cell line HepG2/C3A. GNPAT was efficiently knocked down by its siRNA by ~85% compared to control siRNA as assayed by qPCR. This knockdown resulted in a >17-fold decrease in HAMP mRNA expression. mRNA expression of two genes coordinately regulated with HAMP, ID1 (inhibitor of DNA binding protein 1) and SMAD7 (SMAD family member 7), was similarly decreased, as was expression of phospho-SMAD 1/5/8, suggesting that GNPAT knockdown affects the baseline activity of the bone morphogenetic protein 6 (BMP6)-SMAD pathway. Our data indicate that GNPAT p.D519G is associated with a high-iron phenotype in male HFE C282Y homozygotes and may participate in hepcidin regulation, thereby modifying severity of iron overload. The results identify GNPAT as a candidate gene for expanded studies to examine its function in regulating iron absorption and metabolism and to identify newly-diagnosed C282Y homozygotes whose risk for development of severe iron overload is great. Disclosures No relevant conflicts of interest to declare.

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