Precise O-Glycosylation Site Localization of CD24Fc by LC-MS Workflows.

CD24Fc is a homodimeric recombinant Fc-fusion protein comprised of human CD24 connected to immunoglobulin G1 (IgG1) Fc fragment. CD24 is heavily glycosylated, and its biological function is considered mainly mediated by its glycosylation. Identification of the O-glycosylation sites would facilitate an in-depth understanding of the functional role of O-glycans in CD24. However, the presence of clustered mucin-type O-glycans together with N-glycans makes the determination of O-glycosylation sites and abundance very challenging. In this study, two sets of liquid chromatography-mass spectrometry (LC-MS) workflows were developed for the comprehensive characterization of O-glycosylation in CD24: (1) Fractionation and collision-induced dissociation (CID) workflow involving multienzyme digestion, fractionation, OpeRATOR/SialEXO digestion, and CID analysis; (2) Direct OpeRATOR/SialEXO digestion followed by electron-transfer/higher-energy collision dissociation (EThcD) analysis. The precise O-glycosylation sites were identified in CD24 for the first time, and the site occupancy was assessed. A total of 12 O-glycosylation sites were identified. Seven glycosylation sites were identified by both workflows, and five additional sites were identified only by the EThcD workflow. The predominant O-glycosylation site in CD24 was Thr25 followed by Thr15. The CID workflow provided an overall relative quantitation of O-glycoforms at the CD24 level and site localization for singly O-glycosylated peptides. The EThcD workflow directly identified glycosylation sites by tandem mass spectrometry (MS/MS) for singly, doubly, and triply O-glycosylated peptides. Together, both workflows validated each other's results and can be applied to a complex mucin-type O-glycosylation site analysis of other glycoproteins and Fc-fusion therapeutics.

Structure and activity of human surfactant protein D from different natural sources.

Surfactant protein D (SP-D) is a C-type lectin that participates in the innate immune defense of lungs. It binds pathogens through its carbohydrate recognition domain in a calcium-dependent manner. Human surfactant protein D (hSP-D) has been routinely obtained from bronchoalveolar lavage of patients suffering from pulmonary alveolar proteinosis (PAP) and from amniotic fluid (AF). As a consequence of the disease, hSP-D obtained from PAP is found in higher amounts and is mainly composed of higher order oligomeric forms. However, PAP-hSP-D has never been directly compared with nonpathological human protein in terms of structure and biological activity. Moreover, the quantitative distribution of the different hSP-D oligomeric forms in human protein obtained from a natural source has never been evaluated. In this work, we have determined the quantitative distribution of AF-hSP-D oligomers, characterized the sugars attached through the N-glycosylation site of the protein, and compared the activity of hSP-D from AF and PAP with respect to their ability to bind and agglutinate bacteria. We have found that fuzzy balls (40%) are the most abundant oligomeric form in AF-hSP-D, very closely followed by dodecamers (33%), with both together constituting 73% of the protein mass. The glycan attached to the N-glycosylation site was found to be composed of fucose, galactose, sialic acid, and N-acetylglucosamine. Finally, in the functional assays performed, hSP-D obtained from PAP showed higher potency, probably as a consequence of its higher proportion of large oligomers compared with hSP-D from AF.

Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks.

BACKGROUND: Here we describe a novel cultivation method, called EnBasetrade mark, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and sub-millilitre scale which yields 5 to 20 times higher cell densities compared to standard methods. The novel method can be directly applied in microwell plates and shake flasks without any requirements for additional sensors or liquid supply systems. EnBase is therefore readily applicable for many high throughput applications, such as DNA production for genome sequencing, optimisation of protein expression, production of proteins for structural genomics, bioprocess development, and screening of enzyme and metagenomic libraries. RESULTS: High cell densities with EnBase are obtained by applying the concept of glucose-limited fed-batch cultivation which is commonly used in industrial processes. The major difference of the novel method is that no external glucose feed is required, but glucose is released into the growth medium by enzymatic degradation of starch. To cope with the high levels of starch necessary for high cell density cultivation, starch is supplied to the growing culture suspension by continuous diffusion from a storage gel.Our results show that the controlled enzyme-based supply of glucose allows a glucose-limited growth to high cell densities of OD600 = 20 to 30 (corresponding to 6 to 9 g l-1 cell dry weight) without the external feed of additional compounds in shake flasks and 96-well plates. The final cell density can be further increased by addition of extra nitrogen during the cultivation. Production of a heterologous triosphosphate isomerase in E. coli BL21(DE3) resulted in 10 times higher volumetric product yield and a higher ratio of soluble to insoluble product when compared to the conventional production method. CONCLUSION: The novel EnBase method is robust and simple-to-apply for high cell density cultivation in shake flasks and microwell plates. The potential of the system is that the microbial growth rate and oxygen consumption can be simply controlled by the amount (and principally also by the activity) of the starch-degrading enzyme. This solves the problems of uncontrolled growth, oxygen limitation, and severe pH drop in shaken cultures. In parallel the method provides the basis for enhanced cell densities. The feasibility of the new method has been shown for 96-well plates and shake flasks and we believe that it can easily be adapted to different microwell and deepwell plate formats and shake flasks. Therefore EnBase will be a helpful tool especially in high throughput applications.

Human chorionic gonadotropin (hCG) as inhibitior of E-selectin-mediated cell adhesion.

OBJECTIVE: Human chorionic gonadotropin (hCG) is a placental glycoprotein hormone of clinical importance. It is secreted into maternal circulation, amniotic fluid, urine and is also found in the serum and urine of patients with malignant trophoblastic diseases. Depending on the source, we detected a remarkable expression of sialyl Lewisx on this protein. Sialyl Lewis' is known as a minimal ligand recognized by selectins, which are involved in cell adhesion processes in inflammatory and metastatic diseases. In this context, the purpose of the study was to explore the ability of hCG to serve as a selectin antagonist. MATERIALS AND METHODS: We purified hCG from human amniotic fluid, serum and urine of pregnant women and from supernatants of the trophoblastic tumour cell lines Jeg3 and BeWo by immunoadsorption chromatography. The proteins were functionally tested for a specific blockade of the E-selectin-mediated cell adhesion in vitro. The efficiencies of hCG from different origins were compared. RESULTS: We found that hCG isolated from serum, amniotic fluid and supernatant of the cell line Jeg3 were strong inhibitors with at least 10(3)-fold higher potency compared to the monovalent tetrasaccharide sialyl Lewisx. As expected from the carbohydrate expression, hCG isolated from the urine of pregnant women, but even from the supernatant of BeWo cells, showed no inhibitory effect. CONCLUSION: hCG is an effective selectin antagonist. This fact suggests that hCG may play a role in preventing leukocyte adhesion on the foetal syncytiotrophoblast or the metastatic activity of trophoblastic tumour cells.