EXPRESSION OF ENDOGENOUS RECEPTORS FOR NEOGLYCOPROTEINS, ESPECIALLY LECTINS, THAT ALLOW FIBER TYPING ON FORMALDEHYDE-FIXED, PARAFFIN-EMBEDDED MUSCLE BIOPSY SPECIMENS - A GLYCOHISTOCHEMICAL, IMMUNOHISTOCHEMICAL, AND GLYCOBIOCHEMICAL STUDY
EXPRESSION OF ENDOGENOUS RECEPTORS FOR NEOGLYCOPROTEINS, ESPECIALLY LECTINS, THAT ALLOW FIBER TYPING ON FORMALDEHYDE-FIXED, PARAFFIN-EMBEDDED MUSCLE BIOPSY SPECIMENS - A GLYCOHISTOCHEMICAL, IMMUNOHISTOCHEMICAL, AND GLYCOBIOCHEMICAL STUDY
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DOI：
10.1177/37.7.2732460
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发表时间：
1989-07-01
影响因子：
3.2
通讯作者：
GABIUS, HJ
中科院分区：
文献类型：
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作者：
BARDOSI, A;DIMITRI, T;GABIUS, HJ
一组生物素化(Neo)糖蛋白被用来特异性地检测甲醛固定的石蜡包埋的人三角肌、股四头肌、二头肌、大鼠的胫骨和股四头肌以及牛咬肌的内源性糖受体，特别是凝集素。所用的糖组织化学探针由标记的组织化学惰性载体蛋白和各种共价连接的组织化学关键糖段的结合物组成。检测到α-L岩藻糖苷、β-D-半乳糖苷、β-D-木糖苷和α-D-甘露糖苷与肌肉切片的特异性结合，没有物种特异性差异。用糖组织化学方法证明了天然糖结合物中存在N-乙酰化糖的受体，也存在带有磷酸基团的糖的受体，即甘露糖-6-磷酸和半乳糖-6-磷酸。然而，这些结合特异性揭示了物种特异性的差异，例如，大鼠肌肉中缺乏N-乙酰-D-氨基半乳糖特异性受体或半乳糖-6-磷酸特异性受体。其他带电的糖包括葡萄糖醛酸和唾液酸，它们只与牛和大鼠的肌肉结合，或者没有揭示它们在所有类型的肌肉中的受体。这种不同程度的阴离子探针染色可作为进一步确定碳水化合物结合特异性的对照。肌节内仅在A带水平出现阳性糖组织化学反应。在肌原纤维之间的肌浆和肌膜下区域也观察到颗粒状染色。在1型、2A型和2B型纤维中，糖组织化学可检测到的糖受体的表达存在差异。用免疫组织化学和生化方法推测了一种糖组织化学可检测到的糖受体的分子性质。来自肌肉组织的针对内源性β-半乳糖苷特异性凝集素的抗血清将该凝集素定位在与(NEO)糖蛋白一致相似的切片中，检测β-半乳糖苷特异性受体(S)。这种结合模式的相似性有力地支持了这样的假设，即末端带有β-半乳糖苷的(Neo)糖蛋白确实与各自的内源性凝集素结合。凝集素特异性抗血清使我们能够确定糖组织化学纤维分型与酶组织化学分型相对应。此外，使用亲和层析的生化纯化和随后的亲和洗脱只显示免疫组织化学可检测到的β-半乳糖苷特异性凝集素。因此，当冰冻切片无法用于组织化学分析时，使用一组新糖蛋白可以作为甲醛固定的石蜡包埋活检标本准确纤维分型的基础，从而有助于诊断神经肌肉疾病。
A panel of biotinylated (neo)glycoproteins was used for specific detection of endogenous sugar receptors, especially lectins, in formaldehyde-fixed, paraffin-embedded muscle biopsy specimens from human deltoid, quadriceps, and biceps muscles, tibial and quadriceps muscles of rat, and bovine masseter muscle. The glycohistochemical probes used consisted of conjugates of a labeled, histochemically inert carrier protein and various covalently linked, histochemically crucial sugar moieties. Specific binding of .alpha.-L-fucoside, .beta.-D-galactoside, .beta.-D-xyloside, and .alpha.-D-mannoside to muscle sections was detected, showing no species-specific differences. The presence of receptors for the N-acetylated sugars in natural glycoconjugates, and for sugars with a phosphate group, i.e., mannose-6-phosphate and galactose-6-phosphate, was demonstrated glycohistochemically. However, these binding specificities revealed species-specific differences, e.g., the absence of N-acetyl-D-galactosamine-specific receptors or galactose-6-phosphate-specific receptors in rat muscle. Other charged sugars included glucuronic acid and sialic acid, whcih bound only to ox and rat muscle or failed to reveal their respective receptors in all types of muscle investigated. This different extent of staining with anionic probes served as a further control to ascertain carbohydrate binding specificity. Positive glycohistochemical reaction developed within sarcomeres only at the level of A-bands. Granular staining was observed in the sarcoplasm among the myofibrils and also in the subsarcolemmal regions. Differences in expression of glycohistochemically detectable sugar receptors were noted between type 1, type 2A, and type 2B fibers. The molecular properties of one type of glycohistochemically detectable sugar receptor were inferred both immunohistochemically and biochemically. An antiserum against an endogenous .beta.-galactoside-specific lectin from muscle tissue localized this lectin within sections consistently similar to (neo)glycoproteins, detecting .beta.-galactoside-specific receptor(s). This similarity of binding patterns strongly supports the assumptions that (neo)glycoproteins with .beta.-galactoside termini indeed bind to the respective endogenous lectin. The lectin-specific antiserum enabled us to ascertain that glycohistochemical fiber typing corresponds to enzyme histochemical typing. Moreover, biochemical purification using affinity chromatogrpahy and subsequent affinity elution revealed only the immunohistochemically detectable .beta.-galactoside-specific lectin. Consequently, use of a panel of neoglycoproteins, when frozen sections for histochemical analysis are not available, could be a basis for accurate fiber typing on formaldehyde-fixed, paraffin-embedded biopsy specimens, and thus aid in diagnosing neuromuscular disorders.