新型胶原样凝集素的研究进展

胶原样凝集素作为脊椎动物C-型凝集素超家族内的成员,以含有胶原样结构域及糖识别结构域为其显著特征.胶原样凝集素除了“经典”成员甘露聚糖结合凝集素(MBL)、表面活性蛋白A(SP-A)和SP-D外,还发现肝胶原样凝集素1(CL-L1)、肾胶原样凝集素1(CL-K1)和胎盘胶原样凝集素1(CL-P1).在机体固有免疫系统中,这些“新”型胶原样凝集素除了同“经典”胶原样凝集素一样发挥着十分重要的作用外,它们还具有其独特的生理功能.本文综述了这些新发现的胶原样凝集素的分子结构、组织分布以及生物学功能等方面的研究进展.     

C型凝集素的免疫功能

哺乳类Ｃ型凝集素是一个超家族，根据其糖识别域的一级结构分为６个家族，每一家族的成员具有类似的分子结构和生物学功能。其中胶原凝素、选凝素、Ⅱ型淋巴细胞受体及巨噬细胞甘露糖受体等４个家族分子在抗感染、抗肿瘤、抗原提呈、免疫调节、移植排斥及炎症反应中起着重要的作用。     

甘露聚糖结合凝集素的分离纯化及其与单糖配体的结合活性鉴定

从羊血清中分离纯化甘露聚糖结合凝集素 (MBL ) ,并研究其与单糖配体的结合活性 ,为进一步研究MBL与病原微生物的识别结合活性提供研究基础。用甘露聚糖 Sepharose 4B亲和柱行亲和层析 ,从羊血清中分离纯化出MBL分子 ,行还原性SDS PAGE测定其分子量。用VII型胶原酶鉴定其胶原样结构。用酶联凝集素试验 (ELLA)鉴定其与单糖配体的结合活性。得到纯化的羊血清MBL分子有两种单体 ,相对分子质量分别为 2 90 0 0和 330 0 0。羊血清MBL具有胶原样结构 ,可以被胶原酶消化。羊血清MBL与辣根过氧化物酶表面糖链中甘露糖的结合受N 乙酰葡糖胺、L 岩藻糖、D 氨基葡糖、N 乙酰氨基半乳糖不同程度的抑制。MBL与单糖配体的结合活性预示了其潜在的与表面富含这些糖基结构的病原微生物的识别结合能力。     

肝素酶与肿瘤转移

肿瘤细胞实现扩散、转移 ,与免疫细胞趋向炎症反应区一样 ,必须首先穿越由细胞外基质和基底膜组成的屏障。该屏障主要由两种成分构成 :一是结构蛋白 ,包括胶原、层粘素、纤维结合素和玻璃体结合素等 ;二是糖氨聚糖 ,其主要成分是硫酸乙酰肝素蛋白多糖 (ｈｅｐａｒａｎｓｕｌｆａｔｅｐｒｏｔｏｇｌｙｃａｎ ,ＨＳＰＧ)。ＨＳＰＧ主要由一个蛋白核心和数个与之共价连接的线性硫酸乙酰肝素 (ｈｅｐａｒａｎｓｕｌｆａｔｅ ,ＨＳ)侧链组成[1] 。起初认为 ,结构蛋白的破坏是肿瘤细胞转移的决定性步骤 ,因此 ,在过去的 10多年中 ,研究兴趣大多集…     

胶凝素(Collectin)

胶凝素是具有胶原样结构的凝集素,属于C型凝集素超家族。已确定的胶凝素家族成员有SP-A、SP-D、胶固素、MBP和CL-43,可能还有新的成员。胶凝素的结构和功能均与补体成分Clq高度同源,主要通过调理吞噬和激活补体系统而发挥其天然抗感染免疫作用。     

甘露聚糖结合凝集素缺损与自身免疫性疾病

甘露聚糖结合凝集素(MBL)系胶原凝集素家族成员,是天然免疫系统中的重要分子。血清MBL浓度受其结构基因第一外显子几个点突变的影响和启动子区多态性的调控。MBL基因突变使其血清浓度降低,除导致调理吞噬缺损外,还与自身免疫性疾病如系统性红斑狼疮、类风湿性关节炎、干燥综合征、皮肌炎、克隆病、动脉炎等有关。     

富血小板纤维蛋白联合胶原膜修复颅顶骨缺损北大核心

背景:目前有部分关于富血小板纤维蛋白、胶原膜应用于引导骨再生技术中促进骨缺损修复的研究,但将二者联合使用与单纯使用胶原膜或富血小板纤维蛋白膜修复骨缺损的对比研究尚未被报道。目的:比较富血小板纤维蛋白与胶原复合膜、单纯富血小板纤维蛋白膜、单纯胶原膜3种膜的引导骨再生能力。方法:取日本大耳兔24只,在颅顶骨建立3个骨缺损区,分别植入复合膜(自体富血小板纤维蛋白膜与胶原膜)、单纯自体富血小板纤维蛋白膜及单纯胶原膜,植入后2,4,8,12周,进行颅顶骨缺损区X射线及组织学观察。结果与结论:①X射线观察:植入后2周:复合膜组缺损区见边缘模糊的密度增高影;胶原膜组见极少量密度增高影像;富血小板纤维蛋白膜组边缘区出现密度增高影像。植入后12周,复合膜组整个缺损区与周围正常骨密度接近;胶原膜组环形密度增高影像区域较植入后8周扩大,但中心的密度较周边正常骨组织密度低;富血小板纤维蛋白膜组缺损区可见个别部位的密度低于周围正常骨;②组织学观察:植入后2周,复合膜组见缺损区周围出现纤维结缔组织、新生毛细血管;富血小板纤维蛋白膜膜组、胶原膜组纤维结缔组织和新生血管均少于复合膜组。植入后12周,复合膜组缺损区出现大量骨细胞,排列整齐,有骨小梁变粗变大,可见成熟骨组织形成,骨质较好;富血小板纤维蛋白膜组可见骨细胞,骨小梁数目增多,但骨形成量较复合膜组少;胶原膜组可见少量新骨形成,有成骨细胞、破骨细胞,有明显骨陷凹;③结果表明:富血小板纤维蛋白与胶原复合膜具有更好的成骨效果。     

胶原凝集素结构与功能的研究

胶原凝集素家族是一组寡聚糖蛋白，包括ＭＢＰ，ＣＬ－４３，ＳＰ－Ａ，ＳＰ－Ｄ及牛胶固素。它们基因结构的相似性表明存在进化关联，并赋予这些蛋白分子独特的立体结构，从而对糖类具有特异识别作用，成为病原体与免疫细胞间的桥梁，在先天免疫中发挥重要作用。     

Galectins/intelectins凝集素家族——固有免疫中重要的模式识别分子

半乳糖凝集素(galectins)和intelectins代表了固有免疫系统中2类重要的模式识别分子,通过结合微生物表面的寡糖结构来识别病原体,并介导机体清除外来抗原的免疫反应。Galectins具有保守的糖识别结构域及对β-半乳糖的亲和性。Intelectins是一种分泌型的可溶性糖蛋白,配体是呋喃半乳糖。2种凝集素在识别特异性糖类分子后,激活固有免疫系统,调节感染、炎症及适应性免疫反应。本文对这2种凝集素家族的分类、结构、固有免疫和模式识别功能以及目前的研究热点予以综述。     

Galectins/intelectins凝集素家族——固有免疫中重要的模式识别分子

半乳糖凝集素(galectins)和intelectins代表了固有免疫系统中2类重要的模式识别分子,通过结合微生物表面的寡糖结构来识别病原体,并介导机体清除外来抗原的免疫反应。Galectins具有保守的糖识别结构域及对β-半乳糖的亲和性。Intelectins是一种分泌型的可溶性糖蛋白,配体是呋喃半乳糖。2种凝集素在识别特异性糖类分子后,激活固有免疫系统,调节感染、炎症及适应性免疫反应。本文对这2种凝集素家族的分类、结构、固有免疫和模式识别功能以及目前的研究热点予以综述。     

The role of ficolins in innate immunity

Ficolins are a group of proteins containing both a collagen-like domain and a fibrinogen-like domain and are found in varying tissues. Ficolins present in sera have a lectin activity toward N-acetylglucosamine through their fibrinogen-like domains. The domain organizations between ficolins and mannose-binding lectin (MBL) are very similar in that both consist of a collagen-like domain and a carbohydrate-binding domain, although their carbohydrate-binding moieties are different. MBL acts as an opsonin and activates complement in association with MBL-associated serine proteases (MASPs) and sMAP, a truncated protein of MASP-2, via the lectin pathway. Like MBL, two types of human serum ficolins, L-ficolin/P35 and H-ficolin, are associated with MASPs and sMAP, and activate the lectin pathway. In addition, L-ficolin/P35 acts as an opsonin. These findings indicate that serum ficolins play an important a role in innate immunity in a similar manner to MBL.     

Ficolins and the lectin complement pathway

Ficolins, found in various tissues, are a group of proteins containing both a collagen‐like and a fibrinogen‐like domain. Recently, it was shown that ficolins present in serum are lectins with a common binding specificity for N‐acetylglucosamine (GlcNAc). The fibrinogen‐like domain is responsible for the carbohydrate binding. Mannose‐binding lectin (MBL) is also a collagenous lectin in serum that is specific for GlcNAc and mannose binding. Its domain organization is similar to that of ficolins, except that MBL has a carbohydrate‐recognition domain instead of a fibrinogen‐like domain. MBL plays a role in innate immunity by acting as an opsonin and activating complement in association with MBL‐associated serine protease (MASP) via the lectin pathway. Investigations of two types of human serum ficolins, ficolin/P35 and Hakata antigen, revealed that they are associated with MASPs and sMAP, a truncated protein of MASP‐2, and that they activate complement. These findings indicate that serum ficolins are structurally and functionally similar to MBL and have the capacity to activate the lectin pathway and thus have a role in innate immunity. This work was supported by Grants‐in‐Aid for Scientific Research (12470079 and 11670328) from the Ministry of Education, Science, Sports and Culture of Japan.     

Comparative study of the human ficolins reveals unique features of Ficolin-3 (Hakata antigen)

The ficolins and mannose-binding lectin (MBL) are collagen-like defence proteins that serve as recognition molecules in lectin complement pathway. Differential features that may indicate diverse functions of these proteins are poorly understood. In this study we compared important biological features of the ficolins and MBL. We investigated the tissue distribution of the FCN1-3 and the MBL2 genes encoding the ficolins and MBL by quantitative PCR. Recombinant proteins were produced and structural and biological characteristics were investigated and compared. Our main findings were that FCN3 mRNA was highly expressed in the liver and lung compared with the other genes revealing the lung as the tissue with the highest FCN3 expression pattern. Ficolin-3 revealed higher complement activating capacity compared with Ficolin-2, MBL and Ficolin-1 and was highly resistant to bacterial collagenase treatment, which is different from the other ficolins and MBL. We discovered several unique properties of Ficolin-3 showing that FCN3 is the most highly expressed gene in liver and lung among the lectin complement pathway initiators. Moreover, Ficolin-3 has a high complement activating potential and is the only collagenase proteolytic resistant molecule among the lectin complement pathway initiators.     

Structural and Functional Overview of the Lectin Complement Pathway: Its Molecular Basis and Physiological Implication

The complement system is an effector mechanism in immunity. It is activated in three ways, the classical, alternative and lectin pathways. The lectin pathway is initiated by the binding of mannose-binding lectin (MBL) or ficolins to carbohydrates on the surfaces of pathogens. In humans, MBL and three types of ficolins (L-ficolin, H-ficolin, and M-ficolin) are present in plasma. Of these lectins, at least, MBL, L-ficolin, and H-ficolin are complexed with three types of MBL-associated serine proteases (MASPs), MASP-1, MASP-2, and MASP-3 and their truncated proteins (MAp44 and sMAP). In the lectin pathway, the lectin–MASP complex (i.e., a complex of lectin, MASPs and their truncated proteins) binds to pathogens, resulting in the activation of C4 and C2 to generate a C3 convertase capable of activating C3. MASP-2 is involved in the activation of C4 and C2. MASP-1 activates C2 and MASP-2. The functions of MASP-3, sMAP, and MAp44 in the lectin pathway remain unknown. MASP-1 and MASP-3 also have a role in the alternative pathway. MBL and ficolins are able to bind to a variety of pathogens depending on their carbohydrate binding specificity, resulting in the activation of the lectin pathway. Deficiencies of the components of the lectin pathway are associated to susceptibility to infection, indicating an important role of the lectin pathway in innate immunity. The lectin-MASP complex is also involved in innate immunity by activating the coagulation system. Recent findings suggest a crucial role of MASP-3 in development.     

Role of ficolin in innate immunity and its molecular basis

Ficolin is a multimeric protein consisting of an N-terminal collagen-like domain and a C-terminal fibrinogen-like domain. The structure is similar to mannose-binding lectin (MBL) and complement C1q owing to the collagen-like stalk. Accumulating data indicate that a key function of ficolin is to recognize the carbohydrate moieties on pathogens as a pattern-recognition molecule. Two or three kinds of ficolin have been identified in each species of mammals. They are similar but with some differences in the expression site, location site, ligand-binding specificity and ability to form complexes with MBL-associated serine proteases (MASPs). Like MBL, some ficolins are serum lectins and can form a complex with MASPs and small MBL-associated protein (sMAP). This complex activates the complement through "the lectin pathway". Our recent study suggests that ficolin acts through two distinct routes: the lectin pathway and a primitive opsonophagocytosis. All these observations suggest that ficolins function in clearance of non-self, based on their location sites and their molecular features.     

The lectin pathway of complement: Advantage or disadvantage in HIV pathogenesis?

The pattern recognition molecules of the lectin complement pathway are important components of the innate immune system with known functions in host–virus interactions. This paper summarizes current knowledge of how these intriguing molecules, including mannose-binding lectin (MBL), Ficolin-1, -2 and -3, and collectin-11 (CL-11) may influence HIV-pathogenesis. It has been demonstrated that MBL is capable of binding and neutralizing HIV and may affect host susceptibility to HIV infection and disease progression. In addition, MBL may cause variations in the host immune response against HIV. Ficolin-1, -2 and -3 and CL-11 could have similar functions in HIV infection as the ficolins have been shown to play a role in other viral infections, and CL-11 resembles MBL and the ficolins in structure and binding capacity.     

The mannan-binding lectin pathway and lung disease in cystic fibrosis--disfunction of mannan-binding lectin-associated serine protease 2 (MASP-2) may be a major modifier

The lectin pathway of complement activation is initiated by mannan-binding lectin (MBL) or the ficolins through the common MBL-associated serine protease-2 (MASP-2). Deficiency of MBL has been associated with poorer outcome in cystic fibrosis (CF). We investigated the MBL pathway further by analysis of the MASP-2 deficiency mutation (D105G) as well as MBL-2 genotypes. Concentrations and genotypes of MASP-2 and MBL in 109 CF patients were correlated to lung function and chronic infections. We describe the first CF patient homozygous for the mutation, a girl with extremely severe lung disease with no other precipitating factors. We suspect total MASP-2 dysfunction to be a major modifier of CF lung disease. However, heterozygosity for the D105G mutation of MASP-2 had no correlation to MBL pathway function or poor lung function. Lung function was higher in the MBL deficiency determining genotypes (XA/YO+YO/YO) than in the other genotypes.     

L-ficolin in children with recurrent respiratory infections

The lectin pathway of complement activation is used by a collectin, mannan-binding lectin (MBL), and two ficolins, L-ficolin and H-ficolin, to opsonize microorganisms for phagocytosis. We published evidence recently that MBL insufficiency is associated with recurrent respiratory infections in childhood. We have now measured serum L-ficolin in 313 respiratory infection patients and 74 healthy control children. L-ficolin concentrations below the lower limit of the control group were found in 6% of the patients (P <0.02) and were associated most strongly with children having co-existing atopic disorders (11%; P=0.002). We suggest that L-ficolin may have a role in protection from microorganisms complicating allergic disease.     

Blood Collection Tubes Influence Serum Ficolin-1 and Ficolin-2 Levels

The ficolins are members of a recently discovered family of host innate opsonins that can activate the lectin pathway of complement. The ficolins bind many ligands, although they are typically described as binding acetylated sugars. Ficolin-1 (M-ficolin) and ficolin-2 (L-ficolin) are known to bind Streptococcus pneumoniae serotypes 19C and 11A, respectively. While studying the binding of ficolins to pneumococci, we found variations in ficolin-2 binding among serum samples collected in different types of blood collection tubes. Plastic tubes, which contain a silica clot activator, yielded sera with reduced ficolin-2 binding and apparent ficolin-2 levels. We found that the silica clot activator eluted from plastic red-top tubes inhibited ficolin-2 ligand binding, while other related proteins, like mannose-binding lectin (MBL) and ficolin-1, were not affected. These tube types did not affect the concentrations of other related opsonins (C1q, MBL, or ficolin-3 [H-ficolin]). Interestingly, we also found that ficolin-1 levels were increased 2- to 3-fold in plastic serum separator tubes compared to the increases in other tube types. These findings have implications for future ficolin-1 and ficolin-2 studies, as proper sample collection and handling are essential.     

Levels of mannan-binding lectin-associated serine protease-2 in healthy individuals

The lectin pathway is part of the innate immune system providing a first line of defence against infections. Mannan-binding lectin (MBL) and ficolins, in complex with MBL-associated serine proteases (MASPs), are capable of activating the complement system, thus mediating the destruction of infectious agents. MASP-2 cleaves C4 and C2 and is thus crucial for the activation of downstream complement components. We present an assay for quantifying total MASP-2 in plasma and serum samples. The assay is a sandwich type assay using a combination of two monoclonal anti-MASP-2 antibodies, one directed against the N-terminal part of MASP-2 and the other against its C-terminal part. Based on a population of Danish blood donors, the average MASP-2 concentration was estimated at 534 (S.D.+/-213) ng per ml of plasma. Characterization of the MASP-2 protein in serum showed high stability at 4 degrees C and at ambient temperature but a rapid decline at 37 degrees C. Gel permeation chromatography (GPC) indicated that all MASP-2 in serum is present in complexes with MBL and ficolins.