Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells

Yang, Xiaojing; Tao, Shujuan; Orlando, Ron; Brockhausen, Inka; Kan, Frederick W. K.

doi:10.1016/j.carres.2012.05.027

Cited by 11 publications

(12 citation statements)

References 44 publications

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“…HEK293 glycopeptides were highly sialylated. Glycan assignments are consistent with those reported previously for the egg- 46, 47 and HEK293-derived proteins 48, 49 . HEK293 derived HA glycans were the largest (see Table 3).…”

Section: Resultssupporting

confidence: 90%

Comparative Glycomics Analysis of Influenza Hemagglutinin (H5N1) Produced in Vaccine Relevant Cell Platforms

Rininger²,

Jarvis

et al. 2013

J. Proteome Res.

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Hemagglutinin (HA) is the major antigen in influenza vaccines and glycosylation is known to influence its antigenicity. Embryonated hen eggs are traditionally used for influenza vaccine production, but vaccines produced in mammalian and insect cells were recently licensed. This raises the concern that vaccines produced with different cell systems might not be equivalent due to differences in their glycosylation patterns. Thus, we developed an analytical method to monitor vaccine glycosylation through a combination of nanoLC/MSE and quantitative MALDI-TOF MS permethylation profiling. We then used this method to examine glycosylation of HA’s from two different influenza H5N1 strains produced in five different platforms, including hen eggs, three different insect cell lines (High Five™, expresSF+® and glycoengineered expresSF+), and a human cell line (HEK293). Our results demonstrated that (1) sequon utilization is not necessarily equivalent in different cell types, (2) there are quantitative and qualitative differences in the overall N-glycosylation patterns and structures produced by different cell types, (3) ~20% of the N-glycans on the HAs produced by High Five™ cells are core α1,3-fucosylated structures, which may be allergenic in humans, and (4) our method can be used to monitor differences in glycosylation during the cellular glycoengineering stages of vaccine development.

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Section: Resultssupporting

confidence: 90%

Comparative Glycomics Analysis of Influenza Hemagglutinin (H5N1) Produced in Vaccine Relevant Cell Platforms

Rininger²,

Jarvis

et al. 2013

J. Proteome Res.

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“…Based on the premise that the immortalized human oviductal cells display phenotype characteristics of primary human oviduct cells and that the former cells express both mRNA transcripts and protein of HuOVGP1 (Ling et al 2005), we employed an alternative approach and compared the biosynthetic pathways of N-and O-glycosylation in HEK293 cells, where rHuOVGP1 is produced, with those of immortalized human oviductal cells. As expected, we found that the two cell lines are very similar in the pathways of N-and O-linked glycosylation (Yang et al 2012). In view of these findings, it is reasonable to assume that the rHuOVGP1 described in this study is very similar to the native HuOVGP1.…”

Section: Discussionsupporting

confidence: 85%

Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction

Zhao¹,

Yang²,

Jia³

et al. 2016

Reproduction

Self Cite

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The mammalian oviduct synthesizes and secretes a major glycoprotein known as oviductin (OVGP1), which has been shown to interact with gametes and early embryos. Here we report the use of recombinant DNA technology to produce, for the first time, the secretory form of human OVGP1 in HEK293 cells. HEK293 colonies stably expressing recombinant human OVGP1 (rHuOVGP1) were established by transfecting cells with an expression vector pCMV6-Entry constructed with OVGP1 cDNA. Large quantities of rHuOVGP1 were obtained from the stably transfected cells using the CELLSPIN cell cultivation system. A two-step purification system was carried out to yield rHuOVGP1 with a purity of >95%. Upon gel electrophoresis, purified rHuOVGP1 showed a single band corresponding to the 120-150 kDa size range of human OVGP1. Mass spectrometric analysis of the purified rHuOVGP1 revealed its identity as human oviductin. Immunofluorescence showed the binding of rHuOVGP1 to different regions of human sperm cell surfaces in various degrees of intensity. Prior treatment of sperm with 1% Triton X-100 altered the immunostaining pattern of rHuOVGP1 with an intense immunostaining over the equatorial segment and post-acrosomal region as well as along the length of the tail. Addition of rHuOVGP1 in the capacitating medium further enhanced tyrosine phosphorylation of sperm proteins in a time-dependent manner. After 4-h incubation in the presence of rHuOVGP1, the number of acrosome-reacted sperm induced by calcium ionophore significantly increased. The successful production of rHuOVGP1 can now facilitate the study of the role of human OVGP1 in fertilization and early embryo development.

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“…The inconsistency in ATP2B2 abundance might hint to a species-specific expression pattern, however, further investigations are needed to validate this. The oviduct specific glycoprotein OVGP1 is known to play a crucial role in sperm-oviduct interaction, fertilization as well as in early embryogenesis (Yang et al 2012). OVGP1 existed as heavily glycosylated form in the oviductal EVs, suggesting its biological activity.…”

Section: Protein Cargo Of Porcine Oviductal Evsmentioning

confidence: 99%

Dynamic profile of EVs in porcine oviductal fluid during the periovulatory period

et al. 2020

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In mammals, around the time of ovulation, the hormonal profile dynamically changes in synchrony with reproductive events occurring in the oviduct, that is, sperm arrival, fertilization, and early embryo development. Extracellular vesicles (EVs) have been recently recognized as key components of the embryonic milieu; however, composition and function of oviductal EVs during this crucial period remains to be further explored. Therefore, we initially characterized EVs from porcine oviductal fluid specifically around the critical ovulation window: that is, estrus (E), late estrus (LE, day of expected ovulation), post ovulation (PO), and additionally diestrus (D). Total EV numbers gradually rose from D to E, LE and PO (P < 0.05), which corresponded to the total EV protein amount (P < 0.05). Strikingly, the mean size of EVs in PO was significantly smaller than in E and LE groups, which also had a lesser proportion of small EVs (P < 0.05). The EV protein cargoes during the periovulatory period were further analyzed by mass spectrometry. Qualitative analysis detected 1118 common proteins, which are most enriched in the cellular component of EVs/exosomes. Hierarchical clustering indicated similar protein profile within the biological replicates, but large discrepancy among stages. Further quantitative analysis discovered 34 and 4 differentially expressed proteins in the comparison between E and PO and in the comparison between E and LE, respectively. The dynamic EV protein profile together with the quick adaption in EV size and quantity suggests that porcine oviductal EV secretion are under the hormonal influence during the estrus cycle.

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Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells

Cited by 11 publications

References 44 publications

Comparative Glycomics Analysis of Influenza Hemagglutinin (H5N1) Produced in Vaccine Relevant Cell Platforms

Comparative Glycomics Analysis of Influenza Hemagglutinin (H5N1) Produced in Vaccine Relevant Cell Platforms

Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction

Dynamic profile of EVs in porcine oviductal fluid during the periovulatory period

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