Twenty-three N-glycans of human complement component 1q analyzed by liquid chromatography-tandem mass spectrometry

김정은 중앙대학교 대학원 2022 국내석사

병원체가 인간의 조직에 침입하는 순간부터 선천면역의 방어기전이 활성화되며, 첫 번째의 반응을 일으키는 것이 바로 보체계 입니다. 보체 시스템 중 세 번째로 작용하는 고전적 경로는 보체 성분(C1)이 항원-항체 복합체에 결합하여 보체 캐스케이드를 활성화합니다. 보체 성분 C1은 C1q, C1r, C1s로 구성되고, 이 중 C1q의 C-말단에 있는 6개의 구형 영역이 가장 먼저 항체-항원 복합체의 IgG 또는 IgM의 Fc영역에 결합합니다. 글리코실화는 글리칸이 생체분자에 부착되는 복잡한 post-translational modifications 중 하나이며 글리칸은 제품 안정성, 반감기, 면역원성, CDC 및 ADCC에 큰 영향을 미칩니다. 그 중 아세틸화된 시알산 (OAc-Neu5Ac)은 시알리다제 활성을 억제하여 반감기를 증가시키고 시알산의 항원성과 친수성을 감소시킵니다. 현재까지 항체의 Fc 영역의 N-글리칸 조성에 따른 C1q의 영향에 대해서는 많이 연구가 되었지만 C1q 자체의 N-글리칸 분석은 많이 연구되지 않았습니다. 본 연구에서는 (1) HPAEC-PAD를 사용한 단당 함량 분석, (2) UPLC-FLR과 HILIC 컬럼을 사용한 N-글리칸 분석, (3) LC-ESI-HCD-MS/MS를 사용한 글리칸 구조 및 상대적 함량 분석을 진행하였습니다. 더욱 자세한 N-글리칸의 구조를 분석하기 위해 PNGase F 효소를 사용해 N-글리칸을 방출시키고 ProA 형광 라벨링으로 감도를 높였습니다. 이 연구에서 총 23개의 C1q의 N-글리칸을 분석하였고, MS/MS 스펙트럼으로 구조 확인과 함량 분석을 진행하였습니다. 23가지의 글리칸 중 시알산화와 퓨코실화된 N-글리칸이 74.47%에 해당하였으며 FA2G2S1 (51.59%), FA2G2S2 (17.20%), A2G2S1 (15.10%)의 3개의 N-글리칸이 C1q내에 가장 많이 함유되어 있었습니다. 추가로, 현재까지 보고되지 않은 종류의 아세틸레이션 된 시알산 (OAc-Neu5Ac)을 가지는 N-글리칸을 규명하였습니다. 이번 연구를 통해 C1q의 단당, 23가지의 N-글리칸 (OAc-Neu5Ac 포함)을 규명하고 상대적 함량 값을 분석하였습니다. 이러한 연구는 보체 캐스캐이드의 활성화에 대한 C1q의 역할 규명과 인간 당단백질 또는 바이오의약품에서 N-글리칸의 분석 방법 적용에 유용할 것입니다. From the moment a pathogen invades human tissue, the defense mechanism of the innate immunity is activated, and the first reaction is the complement system. The classical pathway of the complement system is that C1q, one of the components of the complement component (C1), first binds to the antigen-antibody complex and activates the subsequent complement cascade. Glycosylation is one of the complex post-translational modifications that glycans attached to protein, and glycans have a significant impact on product stability, half-life, immunogenicity, complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity. Most studies have been conducted on the effect of N-glycan composition in the mAb-Fc region on binding to C1q, but the glycans of C1q itself remains poorly understood. In this study, the analysis of monosaccharides in acid-hydrolyzed C1q was analyzed using high-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC-PAD). N-glycans of C1q released by peptide-N-glycosidase enzyme and labeled with procainamide (ProA) was analyzed using ultra-performance liquid chromatography and liquid chromatography-tandem mass spectrometry. As a result, monosaccharide composition was confirmed by HPAEC-PAD analysis, comprising Fuc (2.01%), GlcNAc (10.14%), Gal (29.63%), Glc (43.11%), Man (6.90%), and NeuAc (8.20%). These suggest the presence of N- and O-glycans in C1q. The 23 N-glycans include bi-antennary N-glycans (relative quantity of 94.37% in total N-glycans), which are non-sialylated bi-antennary N-glycan (2.74% in totals), mono-sialylated bi-antennary N-glycan (68.44% in totals), and bi-sialylated bi-antennary N-glycan (23.20% in totals), and tri-antennary N-glycans (5.14% in totals), which are non-sialylated tri-antennary N-glycans (0.09% in totals), mono-sialylated tri-antennary N-glycans (4.92% in totals), and di-sialylated tri-antennary N-glycans (0.13% in totals). Mono-sialylated tetra-antennary N-glycan was the largest N-glycan, although its quantity was only 0.1% of total N-glycans. Additionally, modified N-glycan with O-acetylated sialic acid (OAc-Neu5Ac) was also identified. These results indicate that (1) the presence of N- and O-glycans from monosaccharide analysis, (2) 23 N-glycans with non- to bi-sialylated bi- to tetra-antennary structures, (3) the presence of unusual N-glycan, and (4) their relative quantities in human C1q were firstly identified. These results will be useful to investigate the relationship between the complement system and the antibody.

(The) modulation of host immune responses by porcine circovirus type 2 ORF2 and ORF3

최창용 Graduate School, Korea University 2017 국내박사

Porcine circovirus type 2 (PCV2) is the main etiological agent of postweaning multisystemic wasting syndrome (PMWS). The mechanism of pathogenicity associated with PCV2 infection is still not fully understood. Nevertheless, the fact that large amounts of proinflammatory cytokines within lymphoid tissues are released during the early stage of PCV2 infection may induce chronic inflammatory responses followed by the destruction of lymphoid tissues. However, how PCV2 infection causes an excessive inflammatory response in the host immune system during the early stage of PCV2 infection is still not elucidated. Defining how each ORF of PCV2 manipulates the host immune system may be helpful to understand disease progression of PMWS. In this study, it was found that PCV2 ORF3 directly interact with RGS16 in the cytoplasm of host epithelial cells and its interaction leads to ubiquitin-mediated proteasomal degradation of RGS16. Facilitated degradation of the RGS16 by PCV2 ORF3 further enhances NFB translocation into the nucleus through the ERK 1/2 signaling pathway and increased secretion of IL-6 and IL-8 proinflammatory cytokine. Consequently, more severe inflammatory responses and leukocyte infiltration occur around PCV2 infection site. Furthermore, direct interaction between the PCV2 ORF2 and C1QBP within the cytoplasm of host macrophages was demonstrated in this study. The physical interaction between PCV2 ORF2 and C1QBP inhibits ubiquitin-mediated proteasomal degradation of C1QBP in macrophages. Increased stability of the C1QBP by the interaction with PCV2 ORF2 further enhances phagocytic activity of porcine macrophages through the phosphoinositol-3-kinase (PI3K) signaling pathway. These results may provide the first evidence showing how PCV2 infection facilitates the expression of proinflammatory cytokines, enhances phagocytic activity of macrophages and creates a more severe inflammatory response during the early stages of PCV2 infection.

Gamma subunit of complement component 8 is a neuroinflammation inhibitor : 보체 단백 C8 감마의 신경염증 조절 기능 연구

한진 경북대학교 대학원 2019 국내석사

The complement system is a part of the innate immune system, which consists of a number of small proteins activated by sequential cleavages. The majority of these complement components, such as components 3a (C3a) and C5a, are chemotactic and proinflammatory. However, in this study, we revealed an inhibitory role of complement component 8 gamma (C8G) in neuroinflammation. Novel findings show that the expression level of C8G increases in inflamed brain, and C8G is mainly localized to brain astrocytes. Experiments using recombinant C8G protein and shRNA-mediated knockdown showed that C8G inhibits microglial activation and neuroinflammation. In acute and chronic animal models of Alzheimer's disease (AD), which exhibit strong neuroinflammation, C8G attenuated cognitive decline and glial hyperactivation. In these animals and patients with AD, we found higher C8G levels in brain tissue, cerebrospinal fluid, and plasma. Additionally, we identified sphingosine-1-phosphate receptor 2 (S1PR2) as a novel interaction protein of C8G and demonstrate that astrocyte-derived C8G interacts with S1PR2 to antagonize the proinflammatory action of S1P in microglia. Taken together, our results reveal a previously unrecognized role of complement component C8G as a neuroinflammation inhibitor and indicate that C8G can be used therapeutically for the containment of neuroinflammation and treatment of related neurological diseases, such as AD. 보체 시스템은 선천적인 면역체계의 일부인데, 순차적인 분열(sequential cleavages)에 의해 활성화되는 다수의 작은 단백질로 구성되어 있다. 구성요소 3a(C3a)와 C5a와 같은 이러한 보체요소들의 대부분은 chemotactic하고 전염증성과 같은 성질을 가지고 있다. 본 연구에서는 C8G의 신경 염증 억제 역할을 밝혀냈다. 우리의 새로운 연구결과는 C8G의 발현은 염증이 생긴 뇌에서 발현되며, C8G는 주로 뇌 성상 세포에 발현되고 있음을 보여준다. 재조합 C8G 단백질과 shRNA 매개 녹다운을 이용한 실험은 C8G가 미세아교세포 활성화와 신경 염증을 억제한다는 것을 보여주었다. C8G는 강한 신경 염증을 보이는 알츠하이머병(AD)의 급성 및 만성 동물모델에서 인지 저하와glial hyperactivation를 완화했다. 이러한 AD model 동물들과 AD환자에서, 우리는 뇌 조직, 뇌척수액, 그리고 혈장에서 C8G의 수치가 증가하는 것을 확인했다. 또한, 우리는 스핑고신-1인산염 수용체2(S1PR2)를 C8G의 새로운 상호작용 단백질로 확인하였고, 성상세포에서 유도된 C8G가 S1PR2와 상호 작용하여 미세 글리아의 S1P의 친염증 작용(proinflammatory action)에 경쟁적으로 작용한다는 것을 증명하였다. 종합하면, 우리의 결과는 신경 염증 억제제로서 이전에 입증되지 않았던 보체 성분 C8G의 역할을 밝혀냈고, C8G가 신경 염증의 억제와 AD와 같은 관련 신경학적 질병의 치료에 치료법적으로 사용될 수 있다는 것을 보여준다.