RESUMEN
BACKGROUND: Drosophila melanogaster is a well-studied and highly tractable genetic model system for deciphering the molecular mechanisms underlying various biological processes. Although being one of the most critical post-translational modifications of proteins, the understanding of glycosylation in Drosophila is still lagging behind compared with that of other model organisms. METHODS: In this study, we systematically investigated the site-specific N-glycan profile of Drosophila melanogaster using intact glycopeptide analysis technique. This approach identified the glycans, proteins, and their glycosites in Drosophila, as well as information on site-specific glycosylation, which allowed us to know which glycans are attached to which glycosylation sites. RESULTS: The results showed that the majority of N-glycans in Drosophila were high-mannose type (69.3%), consistent with reports in other insects. Meanwhile, fucosylated N-glycans were also highly abundant (22.7%), and the majority of them were mono-fucosylated. In addition, 24 different sialylated glycans attached with 16 glycoproteins were identified, and these proteins were mainly associated with developmental processes. Gene ontology analysis showed that N-glycosylated proteins in Drosophila were involved in multiple biological processes, such as axon guidance, N-linked glycosylation, cell migration, cell spreading, and tissue development. Interestingly, we found that seven glycosyltransferases and four glycosidases were N-glycosylated, which suggested that N-glycans may play a regulatory role in the synthesis and degradation of N-glycans and glycoproteins. CONCLUSIONS: To our knowledge, this work represents the first comprehensive analysis of site-specific N-glycosylation in Drosophila, thereby providing new perspectives for the understanding of biological functions of glycosylation in insects.
Asunto(s)
Drosophila melanogaster , Glicoproteínas , Animales , Drosophila melanogaster/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Polisacáridos/metabolismo , Glicopéptidos/química , Glicopéptidos/metabolismo , Insectos/metabolismoRESUMEN
Intact glycopeptide identification has long been known as a key and challenging barrier to the comprehensive and accurate understanding the role of glycosylation in an organism. Intact glycopeptide analysis is a blossoming field that has received increasing attention in recent years. MS-based strategies and relative software tools are major drivers that have greatly facilitated the analysis of intact glycopeptides, particularly intact N-glycopeptides. This article provides a systematic review of the intact glycopeptide-identification process using MS data generated in shotgun proteomic experiments, which typically focus on N-glycopeptide analysis. Particular attention is paid to the software tools that have been recently developed in the last decade for the interpretation and quality control of glycopeptide spectra acquired using different MS strategies. The review also provides information about the characteristics and applications of these software tools, discusses their advantages and disadvantages, and concludes with a discussion of outstanding tools.
Asunto(s)
Glicopéptidos/análisis , Programas Informáticos , Animales , Humanos , Espectrometría de Masas , ProteómicaRESUMEN
With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N- and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N- and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc N-glycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi- and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N- and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest.
Asunto(s)
Medios de Cultivo/química , Eritropoyetina , Glicopéptidos/química , Fragmentos Fc de Inmunoglobulinas , Proteínas Recombinantes de Fusión , Animales , Células CHO , Cricetulus , Eritropoyetina/biosíntesis , Eritropoyetina/química , Eritropoyetina/genética , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Sodium butyrate (NaBu) is not only well-known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4-O-Bu3 ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4-O-Bu3 ManNAc on EPO glycan quality, while evident in wild-type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of ß-1,4- and ß-1,6-N-acetylglucosaminyltransferases (GnTIV and GnTV) and α-2,6-sialyltransferase (ST6) enzymes responsible for N-glycan antennarity and sialylation. Supplementation of 1,3,4-O-Bu3 ManNAc achieves approximately 30% sialylation enhancement on EPO protein in wild-type CHO cells. Overexpression of GnTIV/GnTV/ST6 in CHO cells increases EPO sialylation about 40%. Combining 1,3,4-O-Bu3 ManNAc treatment in glyocengineered CHO cells promotes EPO sialylation about 75% relative to EPO from wild-type CHO cells. Moreover, a detailed mass spectrometric ESI-LC-MS/MS characterization of glycans at each of the three N-glycosylation sites of EPO showed that the 1st N-site is highly sialylated and either the negative impact of NaBu or the beneficial effect 1,3,4-O-Bu3 ManNAc treatments mainly affects the 2nd and 3rd N-glycan sites of EPO protein. In summary, these results demonstrate 1,3,4-O-Bu3 ManNAc can compensate for the negative effect of NaBu on EPO glycan quality while simultaneously enhancing recombinant protein yields. In this way, a platform that integrates glycoengineering with metabolic supplementation can result in synergistic improvements in both production and glycosylation in CHO cells.