人胚肺成纤维细胞株纤连蛋白结构域N—糖链结构研究

本文应用明胶、肝素亲和层析二步法首先纯化了人胚肺成纤维细胞培养液的纤连蛋白（Ｆｉｂｒｏｎｅｃｔｉｎ,Ｆｎ）,经ＳＤＳ－ＰＡＧＥ鉴定为一条带,然后用胰糜蛋白酶消化纯化的Ｆｎ所获得的酶解液,经分离分别得到明胶结合片段和肝素结合片段,再应用凝集素－ＨＲＰ染色的Ｗｅｓｔｅｒｎ转移电泳法研究糖链结构,结果证实：１．Ｆｎ中明胶结合片段（４４ｋｄ）中含有二天线和多天线复杂型糖链,并接有平分型ＧｌｃＮＡｃ糖基、核     

人胎盘纤连蛋白N糖链结构研究

分离纯化了人胎盘纤连蛋白（Ｆｎ）,经ＳＤＳ－ＰＡＧＥ鉴定为一条带,纯化Ｆｎ仍保持其搞原性,得率为３８．７％。根据植物凝集素识别专一糖链结构的原理,应用斑点印迹法,亲和层析法和Ｗｅｓｔｅｒｎ转移电泳研究糖链结构,结果证实：１．人胎盘Ｆｎ分子中含有复杂型Ｎ糖链（包括二天线和大于二天线的结构）以及高甘露糖型和／或杂合型Ｎ糖链;复杂型Ｎ糖链中含有平分型ＧｌｃＮＡｃ,糖链末端也可连有唾液酸;２．胰糜蛋白酶水解而获得的明胶结合片段（４４ｋＤ）含有二天线和多天线复杂型糖链,也可接有平分型ＧｌｃＮＡｃ;３．肝素结合片段（３０ｋＤ）以及明胶、肝素均不结合的Ｆｎ片段不含有多天线复杂型Ｎ糖链。     

人胎盘纤维蛋白N糖链结构研究

分离纯化了人胎盘纤连蛋白,经ＳＤＳ－ＰＡＧＥ鉴定为一条带,纯化Ｆｎ仍保持其搞原性,得率为３８．７％。根据植物凝集素识别专一糖链结构的原理,应用斑点印迹法。亲和层析法和Ｗｅｓｔｅｒｎ转移电泳研究糖链结构,结果证实：１．人胎盘Ｆｎ分子中含有复杂Ｎ糖链（包括二天线和大于二天线的结构）以及高甘露糖型和／或杂合型Ｎ糖链;复杂型Ｎ糖糖链中含有平分型ＧｌｃＮＡｃ,糖链末端也可连有唾液酸;２．胰糜蛋白酶水解而获得     

佛波酯能改变细胞表面糖蛋白N－糖链的结构

利用标记Ｎ－糖链的凝集素亲和层析法研究了佛波醇肉桂酸乙酸酯（ＰＭＡ）对人肝癌细胞ＳＭＭＣ－７７２１表面糖蛋白上Ｎ－糖链结构的影响,发现１００ｎｍｏｌ／Ｌ的ＰＭＡ处理５天后,可使细胞表面Ｎ－糖链中高甘露糖型和杂合型以及四天线、Ｃ２Ｃ２,６三天线复杂型的比例增高,而二天线复杂型降低。此结果与我们曾报道的视黄酸（ＲＡ）和双丁配环磷酸腺苷（ｄｂ－ｃＡＭＰ）对该细胞表面Ｎ－糖链的影响相反。因ＲＡ和ｄｂ－ｃＡＭＰ是ＳＭＭＣ－７７２１细胞的分化诱导剂,可抑制细胞生长;而ＰＭＡ是该细胞的增殖促进剂,故细胞表面Ｎ－糖链的变化与细胞的分化和增殖密切相关。     

双丁酰环烯酸腺苷对人肝癌细胞株SMMC—7721表面N—糖…

本文采用系列凝集素柱层析法,并配合外切糖苷酶研究了在双丁酰环磷酸腺苷（ｄＢ－ｃＡＭＰ）作用１－５过程中人肝癌细胞株ＳＭＭＣ－７７２１细胞表面Ｎ－糖链类型及复杂型糖链天线数的变化。结果表明,ｄＢ－ｃＡＭＰ促进＾３Ｈ－Ｍａｎ参人细胞表面Ｎ－糖链,使高甘露糖型Ｎ－糖链的百分比下降,并促进二天线Ｎ－糖链的生物合成,使多天线特别是四天线和Ｃ２Ｃ２Ｃ６三天线Ｎ－糖链的百分比减少。结果提示,Ｎ－糖链结构的这些变     

双丁酰环磷酸腺苷耐人肝癌细胞株SMMC－7721表面N－糟链类型及天线数的影响

本文采用系列凝集素柱层析法,并配合外切精苷酶研究了在双丁酰环磷酸腺苷（ｄＢ－ｃＡＭＰ）作用１～５天过程中人肝癌细胞株ＳＭＭＣ－７７２１细胞表面Ｎ－糖链类型及复杂型糖链天线数的变化。结果表明,ｄＢ－ｃＡＭＰ促进３Ｈ—Ｍａｎ参入细胞表面Ｎ－糖链,使高甘露糖型Ｎ－糖链的百分比下降,并促进二天线Ｎ－糖链的生物合成。使多天线特别是四天线和Ｃ２Ｃ２Ｃ６三天线Ｎ－糖链的百分比减少．结果提示,Ｎ－糖链结构的这些变化可能是ｄＢ－ｃＡＭＰ诱导ＳＭＭＣ－７７２１细胞向正常方向分化的结果。     

佛波酯能改变细胞表面糖蛋白N—糖链的结构

利用标记Ｎ－糖链的凝集素亲和层析法研究了佛波醇肉桂酸乙酸酯对人肝癌细胞ＳＭＭＣ－７７２１表面糖蛋白上Ｎ－糖链结构的影响,发现１００ｎｍｏｌ／Ｌ的ＰＭＡ处理５天后,可使细胞表面Ｎ－糖链中高甘露糖型和杂合型以及四天线,Ｃ２Ｃ２,６三天线复杂型的比例增高,而二天线复杂型降低,此结果与我们曾报道的视黄酸和双丁酰环磷酸腺苷对该细胞表面Ｎ－糖链的影响相反。因ＲＡ和ｄｂ－ｃＡＭＰ是ＳＭＭＣ－７７２１细胞的分化诱     

糖链结构不同的纤连蛋白与HT1080细胞结合研究

人血浆纤连蛋白（Ｆｉｂｒｏｎｅｃｔｉｎ,Ｆｎ）与人胎盘纤连蛋白两者在肽链结构上基本相同,但人血浆Ｆｎ的Ｎ－糖链结构为二天线结构,而人胎盘Ｆｎ不仅Ｎ－糖链的数量增加,同时还含有多天线结构,分别用~（１２５）Ｉ标记这两种具有不同糖链结构的Ｆｎ,观察两者与ＨＴ１０８０细胞的饱和结合的亲和性,结果发现,在４℃,人血浆Ｆｎ与ＨＴ１０８０细胞的饱和结合为１２９ｎｇ／１０~５细胞,解离常数为２．８３×１０~（－８）ｍｏｌ／Ｌ,人胎盘Ｆｎ与ＨＴ１０８０细胞的饱和结合为１３３ｎｇ／１０~６细胞,解离常数为２．６４×１０~（－８）ｍｏｌ／Ｌ．因而,人血浆Ｆｎ与人胎盘Ｆｎ上Ｎ－糖链的不同并未影响其与受体的结合．     

大鼠诱发肝癌过程中N乙酰氨基葡萄糖转移酶V的高效液相层析测定

提纯人血浆运铁蛋白,经链霉蛋白酶水解,再经肼解法制备Ｔｆ中的二天线Ｎ糖链,后者经还原末端的氨基吡啶化进行荧光标记,再切除外链的唾液酸和半乳糖残基,获得Ｇｎ２Ｍａｎ３Ｇｎ２－ＰＡ荧光标记糖链,以此制备的糖链为受体底物,ＵＤＰ－ＧｌｃＮＡｃ为供体底物,用反相ＨＰＬＣ分离底物和产物,建立了Ｎ乙酰氨基葡萄糖转移酶测定法,用ＧｎＴ－Ｖ作为肿瘤生化标志物,观察到在二乙基亚硝胺诱发大鼠肝癌的过程中,此酶在诱癌第     

多天线糖链对运铁蛋白与受体结合及内吞的研究

应用系列凝集素柱层析法（伴刀豆球蛋白,小扁豆凝集素,欧曼陀罗凝集素）分别从正常人血清及孕妇血清中提纯含有二天线无核心岩藻糖复杂型糖链的运铁蛋白及含有多天线无核心岩藻糖复杂型糖链的运铁蛋白,与正常的含有二天线糖链的运铁蛋白相比,含有多天线糖链的运铁蛋白,与正常的含有二天线糖链的运铁蛋白相比,含有多天线糖链的运铁蛋白与ＳＭＭＣ－７７２１细胞膜表面的运铁蛋白受体的亲和力下降,但最大结合量不变,此外,其在     

Heparin-binding fibronectin fragments containing cell-binding domains and devoid of hep2 and gelatin-binding domains promote human embryo fibroblast proliferation.

The proliferation promoting activity of various proteolytic fragments of human plasma fibronectin was assayed. Study of this activity in fragments, purified by affinity chromatography, has shown that only heparin-binding fragments were capable of promoting fibroblast proliferation while gelatin- and fibrin-binding fragments were not. Heparin-binding fragments with high affinity for heparin were characterized by high activity levels while those with low heparin affinity were inactive. Heparin-binding fragments with the highest proliferation promoting activity contained the cell-binding domain and were virtually devoid of the hep2, hep1 and gelatin-binding domains.     

Characterization of a 59 kDa gelatin-binding fragment of buffalo plasma fibronectin

Limited proteolysis of buffalo plasma fibronectin (FN) by thermolysin yielded four gelatin-binding fragments of which, the major 59 kDa fragment, GBF1, was isolated by gelatin-Sepharose and heparin-Sepharose affinity columns. GBF1 appeared during early phase of thermolysin digestion and remained intact even after 4 hr of digestion. GBF1 may be similar to 56 kDa gelatin-binding fragment of FNs from human and hamster plasma. But, it is more resistant to thermolysin cleavage. The fragment binds to heparin with low affinity. On the basis of the structure of human plasma FN, the modular structure of GBF1 may be given as: 6Fn1 1Fn2 2Fn2 7Fn1 8Fn1 9Fn1 1Fn3. Biophysical properties of GBF1 suggest an expanded native conformation. The interaction of the fragment with gelatin is pH-dependent and independent of NaCl concentration.     

Interaction of culture-derived macrophages with the fibroblast-binding domain of fibronectin is a necessary but inefficient signal for fibronectin enhancement of CR1-mediated phagocytosis

Human plasma fibronectin (Fn) enhances ingestion of opsonized particles through its interaction with phagocytic cells. To better characterize the site or sites on Fn responsible for this effect, we subjected Fn to limited proteolytic cleavage by chymotrypsin and used affinity and gel filtration chromatography to isolate a 110,000 dalton cell-binding fragment, a 60,000 dalton fragment that bound both heparin and gelatin, and 50,000 and 45,000 dalton fragments that bound to gelatin but not heparin. The cell-binding fragment mediated adhesion and spreading of fibroblasts on glass slides, whereas the heparin-gelatin and gelatin-binding fragments failed to cause fibroblast spreading. At high concentrations, the cell-binding fragment doubled phagocytosis of C4b-coated sheep erythrocytes by human monocyte-derived macrophages, whereas equal concentrations of the other fragments had minimal enhancing effect on phagocytosis. Interestingly, the effect of the cell-binding fragment on CR1-mediated phagocytosis was always less than the effect of intact Fn, even when a 40-fold higher molar concentration of the cell-binding fragment was used. Fab of a monoclonal anti-Fn, HFn 7.1, which recognizes the 110,000 dalton cell-binding fragment of Fn and inhibits fibroblast binding, blocked enhancement of CR1-mediated phagocytosis by intact Fn. Fab of Fn 8, a monoclonal anti-Fn that binds the heparin-gelatin-binding fragment, failed to inhibit the Fn effect. These data suggest that interaction of the macrophage with the cell-binding domain of Fn is critical for the Fn effect on CR1-mediated phagocytosis. In addition, there may be other domains of the Fn molecule that have a role in augmenting the Fn-phagocyte interaction.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Alignment of biologically active domains in the fibronectin molecule

Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Monoclonal antibody against the N-terminal end of human plasma fibronectin.

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.     

Human placental (fetal) fibronectin: increased glycosylation and higher protease resistance than plasma fibronectin. Presence of polylactosamine glycopeptides and properties of a 44-kilodalton chymotryptic collagen-binding domain: difference from human plasma fibronectin

Human placental fibronectin was isolated from fresh term placenta by urea extraction and purified by gelatin affinity chromatography. A 44-kDa chymotryptic fragment, also purified by gelatin affinity chromatography, gave a broad, diffuse band on polyacrylamide gel electrophoresis, whereas the analogous 43-kDa fragment from human plasma fibronectin migrated as a defined, narrow band. Upon extended treatment with endo-beta-galactosidase from Escherichia freundii, the 44-kDa chymotryptic gelatin-binding fragment from placental fibronectin changed its behavior on gel electrophoresis and migrated as a narrower, more defined band. The carbohydrates on human placental fibronectin contained a large percentage of polylactosamine structures, part of which occurred on the gelatin-binding fragment, comprising almost twice as much carbohydrate as plasma fibronectin. NH2-terminal amino acid sequence analysis of the chymotryptic gelatin-binding fragments from both fibronectins showed the first 21 residues to be identical. Tryptic and chymotryptic peptide maps of the gelatin-binding fragment from placental fibronectin, however, showed differences including several protease-resistant domains not found in the analogous fragment from plasma fibronectin. Intact placental fibronectin contains 20,000 Da of carbohydrate, whereas plasma fibronectin contains 11,000 Da. Placental fibronectin is more protease-resistant than plasma fibronectin, possibly due to the additional carbohydrate. Polyclonal antibodies against either fibronectin completely cross-react with amniotic fluid fibronectin, placental fibronectin, and plasma fibronectin upon Ouchterlony immunodiffusion. Human fibronectins of putatively the same polypeptide structure are, therefore, glycosylated in a dramatically different fashion, depending on the tissue of expression. If the patterns of glycosylation comprise the only difference in the glycoprotein, this may confer the characteristic protease resistance found for each of the fibronectins.     

Fibronectin fibril formation involves cell interactions with two fibronectin domains

Fibronectin fragments and domain-specific antibodies have been used to study the mechanism by which cells reorganize exogenous fibronectin substrata into fibrils. Fibroblasts prevented from protein synthesis, and hence not secreting endogenous fibronectin or other matrix components, reorganized exogenous fibronectin substrata into arrays resembling the matrix of normally cultured cells. Cells also formed fibrils from substrata containing mixtures of cell- and either of two different heparin-binding fibronectin fragments but not from either fragment alone. The gelatin-binding fragment alone or in conjunction with the cell-binding fragment did not promote fibril formation. Antibodies recognizing cell- and either heparin- or the gelatin-binding domains labeled fibrils formed by cells under normal culture conditions or when a substratum of intact fibronectin was used as the sole exogenous source. However, only antibodies recognizing the cell- or either heparin-binding fragment reduced fibrillogenesis from intact fibronectin substrates when added during cell spreading. These data suggest that formation of fibronectin fibrils can occur at the cell surface and that membrane components recognizing the cell- and the heparin-binding domains in fibronectin may cooperate in the assembly process