Hae-Min Park

Liver Extracellular Matrix Providing Dual Functions of Two-Dimensional Substrate Coating and Three-Dimensional Injectable Hydrogel Platform for Liver Tissue Engineering

Decellularization of tissues or organs can provide an efficient strategy for preparing functional scaffolds for tissue engineering. Microstructures of native extracellular matrices and their biochemical compositions can be retained in the decellularized matrices, providing tissue-specific microenvironments for efficient tissue regeneration. Here, we report the versatility of liver extracellular matrix (LEM) that can be used for two-dimensional (2D) coating and three-dimensional (3D) hydrogel platforms for culture and transplantation of primary hepatocytes. Collagen type I (Col I) has typically been used for hepatocyte culture and transplantation. In this study, LEM was compared with Col I in terms of biophysical and mechanical characteristics and biological performance for enhancing cell viability, differentiation, and hepatic functions. Surface properties of LEM coating and mechanical properties and gelation kinetics of LEM hydrogel could be manipulated by adjusting the LEM concentration. In addition, LEM hydrogel exhibited improved elastic properties, rapid gelation, and volume maintenance compared to Col I hydrogel. LEM coating significantly improved hepatocyte functions such as albumin secretion and urea synthesis. More interestingly, LEM coating upregulated hepatic gene expression of human adipose-derived stem cells, indicating enhanced hepatic differentiation of these stem cells. The viability and hepatic functions of primary hepatocytes were also significantly improved in LEM hydrogel compared to Col I hydrogel both in vitro and in vivo. Albumin and hepatocyte transcription factor expression was upregulated in hepatocytes transplanted in LEM hydrogels. In conclusion, LEM can provide functional biomaterial platforms for diverse applications in liver tissue engineering by promoting survival and maturation of hepatocytes and hepatic commitment of stem cells. This study demonstrates the feasibility of decellularized matrix for both 2D coating and 3D hydrogel in liver tissue engineering.

Integrated Glycoproteomics Demonstrates Fucosylated Serum Paraoxonase 1 Alterations in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive type of lung cancer, and the detection of SCLCs at an early stage is necessary for successful therapy and for improving cancer survival rates. Fucosylation is one of the most common glycosylation-based modifications. Increased levels of fucosylation have been reported in a number of pathological conditions, including cancers. In this study, we aimed to identify and validate the aberrant and selective fucosylated glycoproteins in the sera of patients with SCLC. Fucosylated glycoproteins were enriched by the Aleuria aurantia lectin column after serum albumin and IgG depletion. In a narrowed down and comparative data analysis of both label-free proteomics and isobaric peptide-tagging chemistry iTRAQ approaches, the fucosylated glycoproteins were identified as up- or down-regulated in the sera of limited disease and extensive disease stage patients with SCLC. Verification was performed by multiple reaction monitoring-mass spectrometry to select reliable markers. Four fucosylated proteins, APCS, C9, SERPINA4, and PON1, were selected and subsequently validated by hybrid A. aurantia lectin ELISA (HLE) and Western blotting. Compared with Western blotting, the HLE analysis of these four proteins produced more optimal diagnostic values for SCLC. The PON1 protein levels were significantly reduced in the sera of patients with SCLC, whereas the fucosylation levels of PON1 were significantly increased. Fucosylated PON1 exhibited an area under curve of 0.91 for the extensive disease stage by HLE, whereas the PON1 protein levels produced an area under curve of 0.82 by Western blot. The glycan structural analysis of PON1 by MS/MS identified a biantennary fucosylated glycan modification consisting of a core + 2HexNAc + 1Fuc at increased levels in the sera of patients with SCLC. In addition, the PON1 levels were decreased in the sera of the Lewis lung carcinoma lung cancer mouse model that we examined. Our data suggest that fucosylated protein biomarkers, such as PON1, and their fucosylation levels and patterns can serve as diagnostic and prognostic serological markers for SCLC.

Mass spectrometry-based N-linked glycomic profiling as a means for tracking pancreatic cancer metastasis

The aberrant glycosylation profile on the surface of cancer cells has been recognized for its potential diagnostic value towards assessing tumor progression. In this study, we initially investigate N-glycan profiles on the surface of normal (HPDE) and cancerous (Capan-1, Panc-1, and MIA PaCa-2) pancreatic cell lines, which are from different sites of pancreatic tumor. The enzymatically deglycosylated total N-glycans are permethylated via a quantitative solid-phase method and then analyzed by using MALDI-TOF MS and MALDI-QIT-TOF MS. We demonstrate that the level of high-mannose type glycans is higher among Capan-1 cells-pancreatic cancer cells that have metastasized to the liver-than that observed among Panc-1 and MIA PaCa-2 cells-pancreatic cancer cells from the pancreas duct head and tail regions, respectively. Furthermore, the relative abundance of highly-branched sialyted N-glycans is significantly up-regulated on Panc-1 and MIA PaCa-2 pancreatic cancer cells compared to that of normal HPDE pancreas cells. Taken together, these results indicate that specific N-glycosylation profile changes in pancreatic cancer cells can be used to not only distinguish between normal and cancerous cells but also provide more information on their location and metastatic potential.

Detection of Hanganutziu–Deicher antigens in O‐glycans from pig heart tissues by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

In the α1,3‐galactosyltransferase knockout (α‐GalT KO) pig era, identification of the non‐Gal epitopes is necessary for successful pig‐to‐human xenotransplantation. Recently, we successfully detected α‐Gal epitopes as well as Hanganutziu–Deicher (H‐D) antigens from the N‐glycans in the pig heart tissues, which have been considered as promising non‐Gal antigens. However, the profiling of O‐glycan from pig heart tissues had not been performed owing to the difficulty of O‐glycan preparation.

Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG) technique for the comparative and quantitative analysis of total N‐glycans using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N‐glycans using a model glycoprotein (bovine fetuin). Moreover, the i‐QTaG using MALDI‐TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of 13C6/12C6‐2‐aminobenzoic acid‐labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N‐glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N‐glycan peaks from i‐QTaG method showed a good linearity (R2 > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2‐AA labeled N‐glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up‐regulation of the Lewis antigen (∼82%) in sera from prostate cancer patients. In this proof‐of‐concept study, we demonstrated that the i‐QTaG method, which enables to achieve a reliable comparative quantitation of total N‐glycans via MALDI‐TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics.

The sweets standing at the borderline between allo‐ and xenotransplantation

Animal cells are densely covered with glycoconjugates, such as N‐glycan, O‐glycan, and glycosphingolipids, which are important for various biological and immunological events at the cell surface and in the extracellular matrix. Endothelial α‐Gal carbohydrate epitopes (Galα3Gal‐R) expressed on porcine tissue or cell surfaces are such glycoconjugates and directly mediate hyperacute immunological rejection in pig‐to‐human xenotransplantation. Although researchers have been able to develop α1,3‐galactosyltransferase (GalT) gene knockout (KO) pigs, there remain unclarified non‐Gal antigens that prevent xenotransplantation. Based on our expertise in the structural analysis of xenoantigenic carbohydrates, we describe the immunologically significant non‐human carbohydrate antigens, including α‐Gal antigens, analyzed as part of efforts to assess the antigens responsible for hyperacute immunological rejection in pig‐to‐human xenotransplantation. The importance of studying human, pig, and GalT‐KO pig glycoprofiles, and of developing adequate pig‐to‐human glycan databases, is also discussed.

Structural characterization of α-galactosylated O-glycans from miniature pig kidney and endothelial cells

O-Glycan is a major oligosaccharide expressed on the surface of eukaryotic cells that plays a role in immune responses via cell-protein and cell-cell interaction. Unlike N-glycan and glycolipid-derived glycan, O-glycans have not been previously reported in pig organs. Here, we report the first instance of the identification of α1,3 galactosylated O-glycans from pig kidney and endothelial cells by using MALDI-TOF MS and nano-ESI MS/MS. The MALDI profile showed 28 O-glycans from miniature pig kidney and 18 O-glycans from pig endothelial cells. Among them, three potential α-galactosylated glycans considered as immunogenic antigens were identified by CID using ESI-MS/MS; Hexα1,3Hex-HexNAc-HexNAc, Hexα1,3Hex-Hex-HexNAc-HexNAc, and Hexα1,3Hex-Hex-Hex-Hex-HexNAc. This structural information on glycans will provide significant information to clinical studies regarding immune rejection responses induced by carbohydrate antigens during xenotransplantation.

Comparative N-Linked Glycan Analysis of Wild-Type and α1,3-Galactosyltransferase Gene Knock-Out Pig Fibroblasts Using Mass Spectrometry Approaches

Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after α1,3-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (α-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.

Highly sensitive glycosylation analysis of membrane glycoproteins avoiding polymeric contaminants

We established a ‘seize-and-release’ purification method to eliminate polyhexose contaminants for a highly sensitive glycan profiling. Pig liver membrane lysates were pretreated with sodium dodecyl sulfate (SDS) surfactant and subsequently dialyzed to remove polyhexose contaminants. From the purified membrane glycoproteins, glycans were released and identified by mass spectrometry. As a result, we clearly obtained N- and O-glycan profiles of a pig liver, which were not achieved without any pre-treatments. This technique demonstrates a powerful approach for enhancing the sensitivity of MS-based glycan profiling.

The Xeno-glycomics database (XDB): a relational database of qualitative and quantitative pig glycome repertoire

SUMMARY In recent years, the improvement of mass spectrometry-based glycomics techniques (i.e. highly sensitive, quantitative and high-throughput analytical tools) has enabled us to obtain a large dataset of glycans. Here we present a database named Xeno-glycomics database (XDB) that contains cell- or tissue-specific pig glycomes analyzed with mass spectrometry-based techniques, including a comprehensive pig glycan information on chemical structures, mass values, types and relative quantities. It was designed as a user-friendly web-based interface that allows users to query the database according to pig tissue/cell types or glycan masses. This database will contribute in providing qualitative and quantitative information on glycomes characterized from various pig cells/organs in xenotransplantation and might eventually provide new targets in the α1,3-galactosyltransferase gene-knock out pigs era. AVAILABILITY The database can be accessed on the web at http://bioinformatics.snu.ac.kr/xdb.