Most bovine milk fat globule membrane glycoproteins contain asparagine-linked sugar chains with GalNAc beta 1-->4GlcNAc groups.
Most bovine milk fat globule membrane glycoproteins contain asparagine-linked sugar chains with GalNAc beta 1-->4GlcNAc groups.
复制标题
大多数牛乳脂肪球膜糖蛋白含有天冬酰胺连接的糖链，其具有 GalNAc beta 1-->4GlcNAc 基团。
DOI：
10.1093/oxfordjournals.jbchem.a124273
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发表时间：
1993
影响因子：
2.7
通讯作者：
Kobata,A
中科院分区：
文献类型：
--
作者：
Sato,T;Furukawa,K;Greenwalt,DE;Kobata,A
材料和方法牛MFGM糖蛋白的纯化-牛MFGM糖蛋白如绿色walt等人所述纯化。（18）、简而言之，用含0.15 M NaCl（TBS）的20 mM Tris-HCl缓冲液（pH 7.4）中的1% Triton X-114（5 mg蛋白质/ml）提取牛MFGM。在4 ℃下搅拌30 min后，将混合物在100，000 μ g下离心2 h。通过升温至30 ° C并以1，000 μ g离心10分钟对上清液进行相分配。将洗涤剂相用TBS稀释10倍，冷却至4 ° C，并重新分配。用含有0.1%Triton X-100的10 mM Tris-HCl缓冲液（pH 8.0）透析所得去污剂相，并冻干。根据Laemmli（19）的方法，通过每泳道施加400 g蛋白质进行SDS-PAGE和Western印迹-SDS-PAGE;然后将蛋白质转移到硝酸纤维素过滤器（20）。WFA或蓖麻凝集素（RCA）-I的蛋白质印迹分析如下进行。用10 mM Tris-HCl缓冲盐水（pH 7.4）中的1%牛血清白蛋白（BSA）封闭滤膜，用10 μ g/ml辣根过氧化物酶结合的凝集素孵育，然后用500 μ g/ml显色过氧化物酶底物4-氯-1-萘酚孵育（21）。在一些实验中，用BSA封闭的过滤器在150 μ l的0.5 M Tris-HCl缓冲液（pH 8.3）中用5.0单位的N-聚糖酶在37 ℃下处理24小时，或者200 μ g的去唾液酸化的糖蛋白样品在50 μ l的0.3 M柠檬酸盐磷酸盐缓冲液（pH 4.0）中用1.0单位的刀豆多糖-N-乙酰氨基己糖苷酶处理。在SDS-PAGE之前，将含有100 μ g顶孢-半乳糖酸内酯或0.1单位顶孢-N-乙酰氨基半乳糖苷酶的顶孢菌在50 μ l 0.3 M柠檬酸盐磷酸盐缓冲液（pH 4.5）中在37 ℃下处理24小时。从糖蛋白-MFGM糖蛋白（10 mg）中释放Asn-连接的糖链，将其在真空中用P205彻底干燥，如前所述（22）进行肼解15小时。N-乙酰化后，从糖蛋白样品中释放的寡糖混合物的三分之二用NaB 3 H还原，以获得用于结构分析的氚标记的寡糖，剩余的用NaB 2 H还原，以获得用于甲基化分析的氘标记的寡糖。为了便于氘标记的寡糖的检测，加入十分之一的氚标记的寡糖。分析方法-放射性寡糖在吡啶-乙酸盐缓冲液（3：1：387，吡啶：乙酸：水），pH5.4中以70 V/cm进行高压纸电泳90分钟。根据Yamashita等人的报告，通过Bio-Gel P-4柱色谱法对放射性寡糖进行分级。（二十三）.如前所述进行寡糖的甲基化分析（24）。寡糖在固定化凝集素柱上的亲和层析--凝集素柱层析
MATERIALS AND METHODSPurification of Bovine MFGM Glycoproteins-Bovine MFGM glycoproteins were purified as described by Green walt et al.(18). In brief, bovine MFGM was extracted with 1% Triton X-114(5mg protein/ml) in 20mM Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl(TBS). After stirring at 4• Ž for 30min, the mixture was centrifuged at 100,000•~ g for 2 h. The supernatant was subjected to phase partitioning by warming to 30• Ž and centrifugation at 1,000•~ g for 10min. The detergent phase was diluted 10-fold with TBS, cooled to 4• Ž, and repartitioned. The resultant detergent phase was dialyzed against 10mM Tris-HC1 buffer, pH 8.0, containing 0.1% Triton X-100 and lyophilized.SDS-PAGE and Western Blotting-SDS-PAGE was performed according to the method of Laemmli(19) by applying 4ƒÊg protein per lane; proteins were then trans ferred to nitrocellulose filters (20). Western blot analysis with WFA or Ricinus communis agglutinin(RCA)-I was performed as follows. Filters were blockedkedkedwere blocked with 1% bovine serum albumin(BSA) in 10mM Tris-HC1-buffered saline, pH 7.4, incubated with 10ƒÊg/ml lectin conjugated with horseradish peroxidase followed by 500ƒÊg/ml of the chromogenic peroxidase substrate 4-chloro-l-naphthol (21). In some experiments, the filters blocked with BSA were treated with 5.0 units of N-glycanase in 150ƒÊl of 0.5 M Tris-HC1 buffer, pH 8.3, at 37• Ž for 24 h or 200 pg of the desialylated glycoprotein sample was treated with 1.0 unit of jack bean ƒÀ-N-acetylhexosaminidase in 50ƒÊl of 0.3 M citrate phosphate buffer, pH 4.0, containing 100ƒÊg of ƒÁ-galactonolactone or 0.1 unit of Acremonium sp. ƒ¿-N-acetylgalactosaminidase in 50ƒÊ1 of 0.3 M citrate phosphate buffer, pH 4.5, at 37• Ž for 24 h prior to SDS-PAGE. Liberation of Asn-Linked Sugar Chains from Glycopro teins-MFGM glycoproteins(10mg), which were dried thoroughly over P205 in vacuo, were subjected to hydrazin olysis for 15 h as described previously(22). After N-ace tylation, two-thirds of the liberated oligosaccharide mix ture from the glycoprotein sample was reduced with NaB3H, to obtain tritium-labeled oligosaccharides for structural analysis, and the remainder was reduced with NaB2H, to obtain deuterium-labeled oligosaccharides for methylation analysis. To facilitate detection of the deu terium-labeled oligosaccharides, one-tenth of the tritiumlabeled oligosaccharides was added. Analytical Methods-The radioactive oligosaccharides were subjected to high-voltage paper electrophoresis in pyridine-acetate buffer(3: 1: 387, pyridine: acetic acid: water), pH 5.4, at 70V/cm for 90min. Fractionation of the radioactive oligosaccharides by Bio-Gel P-4 column chro matography was performed as reported by Yamashita et al.(23). Methylation analysis of oligosaccharides was conducted as described previously(24). Affinity Chromatography of Oligosaccharides on Immo bilized Lectin Columns-Lectin column chromatograph