Proteomic analysis of N‐glycosylation of human seminal plasma

Seminal plasma is a mixture of secretions from several male accessory glands. The seminal plasma contains many secreted proteins which are important for sperm function and male fertility. In this study, we employed N‐linked glycosylated peptide enrichment, combined with LC–MS/MS analysis, and establish the first large scale N‐linked glycoproteome of human seminal plasma. Combined with the results of five biological replicates, a total of 720 N‐glycosylated sites on 372 proteins were identified. Analysis of variations among five individuals revealed similar compositions of N‐glycosylated proteins in seminal plasma. The N‐linked glycoproteome could help us understanding the biological functions of human seminal plasma. The data set could also be a resource for further screening of biomarkers for male diseases including cancer and infertility at the level of N‐glycosylation. For example, N‐glycosylated prostate‐specific antigen is known to be an efficient biomarker that can distinguish benign prostate hyperplasia from prostate cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD000959 ( http://proteomecentral.proteomexchange.org/dataset/PXD000959 ).     

Rational bioprocess design for human pluripotent stem cell expansion and endoderm differentiation based on cellular dynamics

We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation, and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and, with quantitative cell division tracking and fate monitoring, identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion, during directed differentiation, to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.     

Micro‐ and macroheterogeneity of N‐glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum

Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid‐sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site‐specific characterization of the N‐ and O‐linked glycosylation of serum‐derived hSHBG. MS‐driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom‐up and top‐down manner to determine glycosylation sites, site‐specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum‐derived hSHBG is N‐glycosylated at Asn³⁵¹ and Asn³⁶⁷ with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi‐ and triantennary N‐Glycoforms with lactosamine‐type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N‐Glycoforms of Asn³⁶⁷ were slightly more branched and core fucosylated than Asn³⁵¹ N‐glycoforms due probably to a more surface‐exposed glycosylation site. The N‐terminal Thr⁷ was fully occupied by the two O‐linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N‐acetylneuraminic acid and GalNAc is N‐acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn³⁵¹ glycan occupancies and N‐glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.     

Facile removal of high mannose structures prior to extracting complex type N‐glycans from de‐N‐glycosylated peptides retained by C18 solid phase to allow more efficient glycomic mapping

The relative amount of high mannose structures within an N‐glycomic pool differs from one source to another, but quite often it predominates over the larger size complex type structures carrying biologically important glyco‐epitopes. An efficient method to separate these two classes of N‐glycans would significantly aid in detecting the lower abundant components by MS. Capitalizing on an initial observation that only high mannose type structures were recovered in the flow‐through fraction when peptide‐N‐glycosidase F digested peptides were passed through a C18 cartridge in 0.1% formic acid, we demonstrated here that native complex type N‐glycans can be retained by C18 cartridge and to be efficiently separated from both the smaller high mannose type structures, as well as de‐N‐glycosylated peptides by stepwise elution with increasing ACN concentration. The weak retention of the largely hydrophilic N‐glycans on C18 resin is dependent not only on size but also increased by the presence of α6‐fucosylation. This was shown by comparing the resulting N‐glycomic profiles of the washed and low‐ACN eluted fractions derived from both a human cancer cell line and an insect cell line.     

Ratio‐dependent significance thresholds in reciprocal 15N‐labeling experiments as a robust tool in detection of candidate proteins responding to biological treatment

Metabolic labeling of plant tissues with ¹⁵N has become widely used in plant proteomics. Here, we describe a robust experimental design and data analysis workflow implementing two parallel biological replicate experiments with reciprocal labeling and series of 1:1 control mixtures. Thereby, we are able to unambiguously distinguish (i) inherent biological variation between cultures and (ii) specific responses to a biological treatment. The data analysis workflow is based on first determining the variation between cultures based on ¹⁵N/¹⁴N ratios in independent 1:1 mixtures before biological treatment is applied. In a second step, ratio‐dependent SD is used to define p‐values for significant deviation of protein ratios in the biological experiment from the distribution of protein ratios in the 1:1 mixture. This approach allows defining those proteins showing significant biological response superimposed on the biological variation before treatment. The proposed workflow was applied to a series of experiments, in which changes in composition of detergent resistant membrane domains was analyzed in response to sucrose resupply after carbon starvation. Especially in experiments involving cell culture treatment (starvation) prior to the actual biological stimulus of interest (resupply), a clear distinction between culture to culture variations and biological response is of utmost importance.     

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high‐density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break‐up using botulinum hemagglutinin (HA), which specifically bound with E‐cadherin and disrupted cell–cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E‐cadherin‐mediated cell–cell connections to facilitate the break‐up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10⁶ cells ml^?1 was obtained by aggregate break‐up into small ones, which was three times higher than that with the conventional culture without aggregate break‐up. Therefore, the temporary activity of HA for disrupting E‐cadherin‐mediated cell–cell connection was key to establishing a simple in situ method for hiPSC aggregate break‐up in bioreactors, leading to high cell density in suspension culture.     

Enhanced detection of in‐gel released N‐glycans by MALDI‐TOF‐MS

Many biologically relevant glycoproteins need to be separated on 1D‐ or 2D‐gels prior to analysis and are available in picomole amounts. Therefore, it is important to have optimized methods to unravel the glycome that combine in‐gel digestions with MALDI‐TOF‐MS. In this technical report, we investigated how the detection of in‐gel released N‐glycans could be improved by MALDI‐TOF‐MS. First, an AnchorChip target was tested and compared to ground steel target using several reference oligosaccharides. The highest signals were obtained with an AnchorChip target and D‐arabinosazone as the matrix; a LOD of 1.3 to 10 fmol was attained. Then, the effect of octyl‐β‐glucopyranoside, a nonionic detergent, was studied during in‐gel peptide‐N⁴‐(acetyl‐ß‐glucosaminyl) asparagine amidase F digestion of standard glycoproteins and during glycan extraction. Octyl‐β‐glucopyranoside increased the intensity and the amount of detected neutral as well as acidic N‐glycans. A LOD of under 7 pmol glycoprotein could be achieved.     

N‐acetylcysteine prevents oxidized low‐density lipoprotein‐induced reduction of MG53 and enhances MG53 protective effect on bone marrow stem cells

MG53 is an important membrane repair protein and partially protects bone marrow multipotent adult progenitor cells (MAPCs) against oxidized low‐density lipoprotein (ox‐LDL). The present study was to test the hypothesis that the limited protective effect of MG53 on MAPCs was due to ox‐LDL‐induced reduction of MG53. MAPCs were cultured with and without ox‐LDL (0‐20 μg/mL) for up to 48 hours with or without MG53 and antioxidant N‐acetylcysteine (NAC). Serum MG53 level was measured in ox‐LDL‐treated mice with or without NAC treatment. Ox‐LDL induced significant membrane damage and substantially impaired MAPC survival with selective inhibition of Akt phosphorylation. NAC treatment effectively prevented ox‐LDL‐induced reduction of Akt phosphorylation without protecting MAPCs against ox‐LDL. While having no effect on Akt phosphorylation, MG53 significantly decreased ox‐LDL‐induced membrane damage and partially improved the survival, proliferation and apoptosis of MAPCs in vitro. Ox‐LDL significantly decreased MG53 level in vitro and serum MG53 level in vivo without changing MG53 clearance. NAC treatment prevented ox‐LDL‐induced MG53 reduction both in vitro and in vivo. Combined NAC and MG53 treatment significantly improved MAPC survival against ox‐LDL. These data suggested that NAC enhanced the protective effect of MG53 on MAPCs against ox‐LDL through preventing ox‐LDL‐induced reduction of MG53.     

N‐acetyl‐L‐cysteine inhibits bleomycin induced apoptosis in malignant testicular germ cell tumors

Antioxidants may prevent apoptosis of cancer cells via inhibiting reactive oxygen species (ROS). However, to date no study has been carried out to elucidate the effects of strong antioxidant N‐acetylcysteine (NAC) on Bleomycin induced apoptosis in human testicular cancer (NTERA‐2, NT2) cells. For this reason, we studied the effects of Bleomycin and NAC alone and in combination on apoptotic signaling pathways in NT2 cell line. We determined the cytotoxic effect of bleomycin on NT2 cells and measured apoptosis markers such as Caspase‐3, ‐8, ‐9 activities and Bcl‐2, Bax, Cyt‐c, Annexin V‐FTIC and PI levels in NT2 cells incubated with different agents for 24 h. Early apoptosis was determined using FACS assay. We found half of the lethal dose (LD₅₀) of Bleomycin on NT2 cell viability as 400, 100, and 20 µg/ml after incubations for 24, 48, and 72 h, respectively. Incubation with bleomycin (LD₅₀) and H₂O₂ for 24 h increased Caspase‐3, ‐8, ‐9 activities, Cyt‐c and Bax levels and decreased Bcl‐2 levels. The concurrent incubation of NT2 cells with bleomycin/H₂O₂ and NAC (5 mM) for 24 h abolished bleomycin/H₂O₂‐dependent increases in Caspase‐3, ‐8, ‐9 activities, Bax and Cyt‐c levels and bleomycin/H₂O₂‐dependent decrease in Bcl‐2 level. Our results indicate that bleomycin/H₂O₂ induce apoptosis in NT2 cells by activating mitochondrial pathway of apoptosis, while NAC diminishes bleomycin/H₂O₂ induced apoptosis. We conclude that NAC has antagonistic effects on Bleomycin‐induced apoptosis in NT2 cells and causes resistance to apoptosis which is not a desired effect in eliminating cancer cells. J. Cell. Biochem. 114: 1685–1694, 2013. © 2013 Wiley Periodicals, Inc.     

Heat shock protein 70 induction by valproic acid delays photoreceptor cell death by N‐methyl‐N‐nitrosourea in mice

Retinal degenerative diseases (RDs) are a group of inherited diseases characterized by the loss of photoreceptor cells. Selective photoreceptor loss can be induced in mice by an intraperitoneal injection of N‐methyl‐N‐nitrosourea (MNU) and, because of its selectivity, this model is widely used to study the mechanism of RDs. Although it is known that calcium‐calpain activation and lipid peroxidation are involved in the initiation of cell death, the precise mechanisms of this process remain unknown. Heat shock protein 70 (HSP70) has been shown to function as a chaperone molecule to protect cells against environmental and physiological stresses. In this study, we investigated the role of HSP70 on photoreceptor cell death in mice. HSP70 induction by valproic acid, a histone deacetylase inhibitor, attenuated the photoreceptor cell death by MNU through inhibition of apoptotic caspase signals. Furthermore, HSP70 itself was rapidly and calpain‐dependently cleaved after MNU treatment. Therefore, HSP70 induction by valproic acid was dually effective against MNU‐induced photoreceptor cell loss as a result of its anti‐apoptotic actions and its ability to prevent HSP70 degradation. These findings might help lead us to a better understanding of the pathogenic mechanism of RDs.

     

ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons

Induced pluripotent stem cells (iPSCs) hold promise to revolutionize studies of intracellular transport in live human neurons and to shed new light on the role of dysfunctional transport in neurodegenerative disorders. Here, we describe an approach for live imaging of axonal and dendritic transport in iPSC‐derived cortical neurons. We use transfection and transient expression of genetically‐encoded fluorescent markers to characterize the motility of Rab‐positive vesicles, including early, late and recycling endosomes, as well as autophagosomes and mitochondria in iPSC‐derived neurons. Comparing transport parameters of these organelles with data from primary rat hippocampal neurons, we uncover remarkable similarities. In addition, we generated lysosomal‐associated membrane protein 1 (LAMP1)‐enhanced green fluorescent protein (EGFP) knock‐in iPSCs and show that knock‐in neurons can be used to study the transport of endogenously labeled vesicles, as a parallel approach to the transient overexpression of fluorescently labeled organelle markers.     

Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patient specific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.     

Microencapsulation of dopamine neurons derived from human induced pluripotent stem cells

Background

Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress.

Methods

hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons.

Results

Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved.

Conclusion

hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads.

General significance

Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons.