Abstract Angiopoietin-1 (Ang1), a potential growth factor for therapeutic angiogenesis and vascular stabilization, is known to specifically cluster and activate Tie2 in high oligomeric forms, which is a unique and essential process in this ligand-receptor interaction. However, highly oligomeric native Ang1 and Ang1 variants are difficult to produce, purify and store in a stable and active form. To overcome these limitations, we developed a simple and active dimeric CMP-Ang1 by replacing the N-terminal of native Ang1 with the coiled-coil domain of cartilage matrix protein (CMP) bearing mutations in its cysteine residues. This dimeric CMP-Ang1 effectively increased the migration, survival and tube formation of endothelial cells via Tie2 activation. Furthermore, dimeric CMP-Ang1 induced angiogenesis and suppressed vascular leakage in vivo . Despite its dimeric structure, the potencies of such Tie2-activation-induced effects were comparable to those of a previously engineered protein, COMP-Ang1. We also revealed that these effects of dimeric CMP-Ang1 were affected by specified N-glycosylation in its fibrinogen-like domain. Taken together, our results indicate that dimeric CMP-Ang1 is capable of activating Tie2 and stimulating angiogenesis in N-glycan dependent manner.
Glycosylation plays important roles m a therapeutic glycoprotein such as stability, efficiency, and pharmacokinetics. Especially, sialic acid expressed as an outer terminal unit on the glycoprotein is involved in half-life in serum. In particular, Neu5Gc, a non-human sialic acid, is often observed in therapeutic glycoproteins produced from mammalian cell lines. And exogenous Neu5Gc can be an immunogenic antigen in human cells. Therefore, the screening and quantitation of Neu5Gc in a therapeutic glycoprotein is highly required in the manufacturing and development processes. In this study, we developed an analytical method for selectively identifying and quantifying Neu5Gc with high sensitvity usmg mass spectrometry. The amount of Neu5Gc isolated from various mAb-based therapeutics such as infliximab and trastuzumab was correctly determined. Briefly, sialic acids were liberated from each therapeutic glycoprotein by chmical hydrolysis and further enriched using solid phases extraction with a PGC cartridge. The Neu5Gc was chromatographically separated on a PGC column, then analyzed by MRM-MS. The concentration of Neu5Gc from mAb-based therapeutics was determined at low nano mole levels with high reproducibility ( CV < 6%). Batch-to-batch variations of Neu5Gc from a therapeutic glycoprotein were quatitatively determined. This method will be a valuable platform for not only QbD processes of biotherapeutics but also QA/QC of products to assess impurites and immunogenecities
Gangliosides in the brain play a crucial role in modulating the integrity of vertebrate central nervous system in a region-specific manner. However, to date, a comprehensive structural elucidation of complex intact ganglioside isomers has not been achieved, resulting in the elusiveness into related molecular mechanism. Here, we present a glycolipidomic approach for isomer-specific and brain region-specific profiling of the mouse brain. Considerable region-specificity and commonality in specific group of regions are highlighted. Notably, we observe a similarity in the abundance of major isomers, GD1a and GD1b, within certain regions, which provides significant biological implications with interpretation through the lens of a theoretical retrosynthetic state-transition network. Furthermore, A glycocentric-omics approaches using gangliosides and N-glycans reveal a remarkable convergence in spatial dynamics, providing valuable insight into molecular interaction network. Collectively, this study uncovers the spatial dynamics of intact glyco-conjugates in the brain, which are relevant to regional function and accelerates the discovery of potential therapeutic targets for brain diseases.
// Jae-Han Kim 1 , Sung Hyeon Lee 2 , Sookyung Choi 2 , Unyong Kim 3 , In Seok Yeo 4 , Su Hee Kim 2 , Myung Jin Oh 3 , Hantae Moon 3 , Jua Lee 3 , Seunghyup Jeong 3 , Min Gew Choi 5 , Jun Ho Lee 5 , Tae Sung Sohn 5 , Jae Moon Bae 5 , Sung Kim 5 , Yang Won Min 5 , Hyuk Lee 5 , Jun Haeng Lee 5 , Poong-Lyul Rhee 5 , Jae J. Kim 5 , Su Jin Lee 5 , Seung Tae Kim 5 , Jeeyun Lee 5 , Se Hoon Park 5 , Joon Oh Park 5 , Young Suk Park 5 , Ho Yeong Lim 5 , Won Ki Kang 5 , Hyun Joo An 3 , Jung Hoe Kim 4 1 Department of Food and Nutrition, Chungnam National University, Yuseong-Gu, Deajeon, Korea 2 GLYCAN Co., Ltd., Healthcare Innovation Park, Bundang-Gu, Seongnam, Korea 3 Graduate School of Analytical Science and Technology, Chungnam National University, Yuseong-Gu, Deajeon, Korea 4 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yuseong-Gu, Daejeon, Korea 5 Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Correspondence to: Se Hoon Park, email: hematoma@skku.edu Keywords: stomach neoplasm, haptoglobin, N-glycan, glycoprotein, biomarker Received: October 05, 2016 Accepted: December 21, 2016 Published: December 29, 2016 ABSTRACT Based on our previous studies, differential analysis of N-glycan expression bound on serum haptoglobin reveals the quantitative variation on gastric cancer patients. In this prospective case-control study, we explore the clinically relevant glycan markers for gastric cancer diagnosis. Serum samples were collected from patients with gastric cancer ( n = 44) and healthy control ( n = 44). N-glycans alteration was monitored by intact analysis of Hp using liquid chromatography–mass spectrometry followed by immunoaffinity purification with the serum samples. Intensity and frequency markers were defined depending on the mass spectrometry data analysis. Multiple markers were found with high diagnostic efficacy. As intensity markers ( I -marker), six markers were discovered with the AUC > 0.8. The high efficiency markers exhibited AUC of 0.93 with a specificity of 86% when the sensitivity was set to 95%. We additionally established frequency marker ( f -marker) panels based on the tendency of high N-glycan expression. The AUC to conclude patients and control group were 0.82 and 0.79, respectively. This study suggested that N-glycan variation of serum haptoglobin were associated with patients with gastric cancer and might be a promising marker for the cancer screening.
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