血清肝型碱性磷酸酶糖链分析方法的改进及其临床应用

本文建立了直接用唾液酸酶切除血清碱性磷酸酶（ＡＬＰ）糖链末端的唾液酸后，用蔓陀萝凝集纱（ＤＳＡ）一支亲和层析柱分析其糖链结构的简单方法，用该法分析了１０例正常人和４９例不同肝病患者血清肝型碱性磷酸酶糖链结构，发现只有肝癌血清ＡＬＰ含有与ＤＳＡ强结合的“多天线（ｍｕｌｔｉａｎｔｅｎｎａｒｙ）结构，这一结果与以臆繁琐的序列凝集亲和层析法所得的结果相同，本文不仅再次证实了ＤＳＡ分析血清ＡＬＰ糖链结构在区     

肝癌组织和肝癌旁组织中γ-谷氨酰转肽酶糖链结构分析

目的比较肝癌组织和肝癌旁组织中γ-谷氨酰转肽酶(γ-GT)活力及其N-连接型糖链结构的变化.方法用荧光分光光度法测定肝癌组织和肝癌旁组织中的γ GT活力,并用固相凝集素亲和层析测定γ-GT的糖链与刀豆球蛋白(ConA)和曼陀罗凝集素(DSA)结合百分比,以研究肝癌γ-GT糖链结构的变化.结果肝癌组织中γGT活力均较肝癌旁组织明显增高.两者相比,肝癌组织中γ GT与ConA柱不结合的组分明显升高,弱结合和强结合的组分显著降低;在DSA柱上则相反,不结合的γ-GT组分显著降低,弱结合和强结合的γ-GT组分显著升高.结论肝组织癌变后,其γ-GT活力明显增高,而且伴随着γ-GT糖链结构的明显变化二天线糖链结构明显减少,三、四天线糖链结构明显增高.提示带多天线糖链结构的γ-GT可能是一种肝癌标志物.     

肝癌组织和肝癌旁组织中γ—谷氨酰转肽酶糖链结构分析

目的：比较肝癌组织和肝癌旁组织中γ－谷氨酰转肽酶（γ-GT）活力及其N－连接型糖链结构的变化。方法：用荧光分光光度法测定肝癌组织和肝癌旁组织中的γ-GT活力，并用固相凝集素亲和层析测定γ-GT的糖链与刀豆球蛋白（ConA）和曼陀罗凝集素（DSA）结合百分比，以研究肝癌γ-GT糖链结构的变化，结果：肝癌组织中γ-GT活力均较肝癌旁组织明显增高，两者相比，肝癌组织中γ-GT与ConA柱不结合的组分明显升高，弱结合和强结合的组分显著降低，在DSA柱上则相反，不结合的γ-GT组分显著降低，弱结合和弱结合的γ-GT组分显著升高，结论：肝组织癌变后，其γ-GT活力明显增高，而且伴随着γ-GT糖链结构的明显变化：二天线糖链结构明显减少，三、四天线糖链结构明显增高，提示带多天线糖链结构的γ-GT可能是一种肝癌标志物。     

肝癌γ-谷氨酰转肽酶糖链的凝集素亲和分析

目的：研究肝癌γ－谷近酰转肽酶（γ－GT）糖链结构的变化以寻找新的肝癌标志物。方法：将肝癌组织和癌旁正常肝组织中γ－GT抽提液、肝癌患者血清和正常人血清，分别用ConA和DSA两种固相凝集素柱进行亲和层析，有荧光分光光度法测定各收集管的γ－GT活力并计算不同结合组分的百分比。结果：肝癌组织与癌旁正常肝组织相比，其γ－GT中与ConA柱不结合的组分明显升高，弱结合和强结合的组分显著降低；在DSA柱上则相反，不结合的γ－GT组分显著降低，弱结合和强结合的γ－GT组分显著升高。血清γ－GT分析结果与此相似。结论：肝组织癌变后，其γ－GT的糖链结构发生了明显变化：2天线糖链结构明显减少，3、4天线糖链结构明显增高。提示带多天线糖链结构的γ－GT可能是一种肝癌标志物。     

肝癌与肝硬化患者血清碱性磷酸酶肝型同工酶的比较

用麦胚凝集素亲和层析柱分离得到血清碱性磷酸酶肝同工酶，再用蔓陀罗凝集素亲和层析柱分析其糖链结构，发现该同工酶在肝癌和肝硬化患者中糖链结构不同，表现为与蔓陀罗凝集素的亲和力不同，肝癌血清含有与蔓陀罗凝集素强结合的肝型同工酶，而这在肝硬化及其它良性肝胆疾病和正常人中均未发现，肝癌血清碱性磷酸酶型同工酶的这种特征的糖链结构，有可能成为肝脏癌变的新的血清学生化标志物。     

肝癌与肝硬化患者血清碱性磷酸酶肝型同工酶的比较

用麦胚凝集素亲和层析柱分离得到血清碱性磷酸酶肝型同工酶，再用蔓陀罗凝集素亲和层析柱分析其糖链结构，发现该同工酶在肝癌和肝硬化患者中糖链结构不同，表现为与蔓陀罗凝集素的亲和力不同。肝癌血清含有与蔓陀罗凝集素强结合的肝型同工酶，而这在肝硬化及其它良性肝胆疾病和正常人中均未发现。肝癌血清碱性磷酸酶肝型同工酶的这种特征的糖链结构，有可能成为肝脏癌变的新的血清学生化标志物。     

应用江苏菜豆红细胞凝集素亲和柱检测肝癌特异γ-谷氨酰移肽酶

由于肝脏γ-谷氨酰移肽酶(GGT)糖链的改变与恶性转化有关。我们利用神经氨酸酶(NMD)消化的人血清GGT 糖链与江苏菜豆红细胞植物血球凝集素(E-PHA)的亲和性差异,区别原发性肝癌和良性肝病。     

亲和吸附法在甲胎蛋白异质体检测中的应用

血清甲胎蛋白(AFP)测定已被广泛应用于肝癌的诊断。人们在发现了AFP存在糖链有差别的异质体后，就利用它们与不同植物凝集素的亲和性进行分类，例如，岩藻糖苷化的AFP亲和豌豆凝集素(LCA)称LCA—AFP—V，氨基葡萄糖化的亲和刀豆素A(ConA)称ConA—AFP—V。许多报道表明测定AFP的某些糖链异质体可明显提高肝癌实验室诊断的特异性和灵敏度。但是长期以来，国内只有少数临床实验室能够测定AFP异质体，     

脂蛋白结合r-谷氨酰转肽酶的测定与肝病诊断

r一谷氨酰转肽酶(EC2、3、2、2)作为一种糖蛋白出现于多种人体组织器官中,在肝胆胰疾病时其血清酶活力可显著升高.可做为一种重要的酶指标应用于肝病及肝癌的诊断中.但只单一测定GGT总活力其诊断特异性不强,目前测定GGT同功酶的方法有特异性底物,热灭活、免疫法、柱层析法和电泳法,其中电泳法应用最广,但需多种仪器设备且操作繁杂.1988年Sacchetti用改良醋酸纤维膜电泳测GGT同功酶同时研究了脂蛋白与GGT同功酶间的关系,认为在肝病时,GGT同功酶可与不同密度的脂蛋白结合,并可用沉淀剂区分,而某些区带及正常人区带则不受影响.本文初步分析了脂蛋白密度沉淀剂对血清GGT的影响,并应用于多种肝病诊断中.     

序列凝集素亲和层析分析人血清骨型ALP糖链结构

用麦胚凝集素（ＷＧＡ）、蔓陀萝凝集素（ＤＳＡ）、伴刀豆蛋白（ＣｏｎＡ）亲和层析技术分析正常小儿和良性骨病患者的血清骨型碱性磷酸酶（ＡＬＰ）的糖链结构，推测出人血清骨型ＡＬＰ的糖链结构主要为：含有平分型Ｎ－乙酰胺基葡萄糖（ｂｉｓｅｃｔｉｎｇ－ＧｌｃＮＡｃ）的“二天线”复杂型Ｎ－糖链，且糖链外侧连有唾液酸（ＳＡ）、不含２，２，６－和２，４，２，６－分枝的“多天线”糖链，为研究良、恶性骨病血清骨型ＡＬＰ     

Tartrate-resistant acid phosphatase 5a and 5b contain distinct sugar moieties

Tartrate-resistant acid phosphatase (TRAP) consists of 2 structurally related isoforms, 5a and 5b, and the latter has been characterized as containing a sugar moiety devoid of sialic acid(s). We provide evidence of a greater difference between the sugar moieties of the two isoforms than the presence of sialic acid(s) in TRAP 5a. TRAP 5a and 5b were highly purified from human blood and femur extracts, respectively, and after purifying the TRAPs by successive chromatography on a series of lectin affinity columns, we used the chromatographs obtained to estimate the composition of their sugar chain structure and found that both TRAP 5a and 5b had contained the heterogenous sugar chains. More specifically, TRAP 5a mainly contained a high-mannose-type sugar chain, while TRAP 5b had multi-antennary complex-type sugar chain. These results indicate that the differences between TRAP 5a and 5b consist not only of a difference in modification by sialic acid but differences in other sugar chain structures. These differences are involved in an extraordinary enzyme maturation process since the carbohydrate chains cover the repressive loop where the proteolytic site is located.     

Significance of acidic sugar chains of apolipoprotein B-100 in cellular metabolism of low-density lipoproteins

We have elucidated the carbohydrate structures of the N-linked sugar chains of human and rabbit apolipoprotein B-100 (apo B-100), which is similar in composition to oligosaccharides (Arch Biochem Biophys 1989;273:197-205, Arteriosclerosis 1990; 10:386-93). We have also shown the negative correlation of the ratio of acidic sugar chains of apo B-100 to the serum cholesterol levels in Watanabe heritable hyperlipidemic rabbits (Atherosclerosis 1992;93:229-35). The acidity of sugar chains is determined by the existence of sialic acid residues at the terminal of oligosaccharides. In the present study we investigated N-linked sugar chains of apo B-100 from patients with coronary artery disease (CAD) who had moderate hypercholesterolemia (less than 400 mg/dL). There was no difference in the structure of their oligosaccharides and the ratio of acidic sugar chains of apo B-100 from CAD patients as compared with that from healthy individuals reported previously. To clarify the role of sialic acid residues in apo B-100 for lipoprotein metabolism, we studied cellular uptake of low-density lipoproteins (LDLs) treated with sialidase (desialylated LDL). Desialylated LDLs were taken up and degraded to a 2-fold greater degree than control LDL by human monocyte-derived macrophages and stimulated cholesterol esterification in these cells. These results indicate that sialic acid residues of apo B- 100 play an important role in cellular uptake and degradation of LDL.     

Differences in the enzymatic nature and the sugar-chain structure of gamma-glutamyl transferase between normal and carcinomatous human kidney and prostate.

The enzymatic and immunological nature, and the sugar chain structure, of gamma-glutamyl transferase (GGT) purified from tissues of benign prostatic hypertrophy (BPH), prostatic carcinoma (PCa) and renal cell carcinoma (RCa), were compared with those of the normal prostate (NP) and kidney (NK). The specific activities of GGTs in NP, NK, BPH, PCa and RCa were 78.9, 22.5, 105, 92.5 and 52.5 mU/mg protein, respectively. The molecular masses of GGTs from BPH, PCa and RCa were 72 kDa, 78 and 108 kDa, and 79 and 105 kDa, respectively. The Michaelis constants (Km), optimum pHs and the inhibition of GGT activities by several chemical compounds, revealed that the GGT from BPH, PCa and RCa was similar to that of normal GGT. Immunologically, the IgG fraction against anti-human seminal plasma GGT fused to the all of the GGTs tested. The sugar chain heterogeneities of the various GGTs, detected by the serial-lectin affinity technique, differed from one another. The sugar chain of GGT from BPH resembled the sugar chain from NP. On the contrary, the sugar chains of GGTs from PCa and RCa were markedly different from those from normal tissues. In the GGT from PCa, multi-antennary complex type sugar chains were more increased than the enzyme of NP. In general, as previously reported, the sugar chains of GGTs from carcinomatous tissues of prostate and kidney had an increased content of bisecting GlcNAc (beta 1-->4) containing complex type sugar chains. Moreover, the reductions of the biantennary complex type sugar chain with fucose linkage and the hybrid type sugar chain were obvious in the GGT from carcinomatous tissues of the prostate and kidney.     

Activation energy and lectin affinity chromatography of gamma-glutamyltransferase as a marker for enzyme heterogeneity

Lectin affinity chromatography of gamma-glutamyl transferase (GGT,EC 2.3.2.2) is able to detect differences in the carbohydrate moiety of the enzyme. Binding of tissue GGT towards lectins is significantly different from serum GGT, showing increased galactosylation in tissue forms. Kidney GGT is less glycosylated than GGT from other tissues (liver, pancreas, prostate, vesiculae seminales). Increases in sialic acid content of GGT are associated with an increase in the activation energy of the catalyzed reaction. Differences in galactose, fucose and N-acetylhexosamine content induce much smaller effects on activation energy. In liver diseases, serum GGT is characterized by an altered affinity against lectins recognizing galactose, fucose and N-acetyglucosamine and by increased activation energy. In patients with liver disease, use of fixed temperature conversion factors can lead to erroneous calculations of serum GGT enzyme activity (errors up to 13.3%).     

Discrimination of gamma-glutamyltranspeptidase from normal and carcinomatous pancreas

Two types of gamma-glutamyltranspeptidase (gamma-GTP) were identified in normal human pancreas, but ductal cell carcinomas of the pancreas showed only one type on analysis with polyacrylamide gel electrophoresis, anion exchange column chromatography, concanavalin A affinity chromatography and isoelectric focusing. gamma-GTP from carcinoma tissue was almost identical to the sialic acid-rich type of normal pancreatic gamma-GTP, and our methods did not evidence a difference between the two. However, E-PHA lectin column chromatography demonstrated differences in their elution pattern. Compared with normal pancreatic gamma-GTPs, the elution pattern of the carcinoma gamma-GTP was slowly delayed by the E-PHA lectin column. This finding suggests the presence of a bisecting N-acetylglucosamine-rich sugar chain in pancreatic carcinoma gamma-GTP, as well as the possible presence of a different sugar structure in the carcinoma gamma-GTP.     

Evidence for an asialoglycoprotein receptor on nonparenchymal cells for O-linked glycoproteins

B cell-activating factor receptor 3 (BR3)-Fc is an IgG1-receptor dimeric fusion protein that has multiple O-linked glycosylation sites and sialylation levels that can vary in the manufacturing process. Increased sialic acid levels resulted from increased site occupancy with the O-linked N-acetylgalactosamine (GalNAc-Gal), but because the ratio of sialic acid per mole of oligosaccharide remained approximately 1, this led to increased asialo terminal GalNAc. Previous studies have demonstrated an effect of terminal asialo Gal or GalNAc on the clearance of glycoproteins due to uptake and degradation by lectin receptors in the liver. However, the previous studies examined N-linked oligosaccharides, and there are less data regarding O-linked oligosaccharides. The objective of these studies was to determine the effects on the pharmacokinetics and distribution of the asialo terminal GalNAc and varying amounts of sialic acid residues on BR3-Fc. The results of the data presented here suggest that exposed Gal on the desialylated BR3-Fc led to rapid clearance due to uptake and degradation in the liver that was associated with nonparenchymal cells. It is interesting to note that the data indicated a decreased clearance and increased exposure of BR3-Fc as the sialic acid levels increased, even though increased sialic acid was associated with increased asialo GalNAc. Therefore, the exposed GalNAc did not seem to play a role in the clearance of BR3-Fc; although the Gal linked to the hydroxyl group at position 3 may have prevented an interaction. Because we did not see uptake of desialylated BR3-Fc in hepatocytes where the asialoglycoprotein receptor is localized, this nonparenchymal cell lectin may have preference for O-linked glycoproteins.     

An N-linked high-mannose type oligosaccharide, expressed at the major outer membrane protein of Chlamydia trachomatis, mediates attachment and infectivity of the microorganism to HeLa cells.

The structure of the carbohydrate of the 40-kD major outer membrane component of Chlamydia trachomatis and its role in defining infectivity of the organism were investigated. The oligosaccharides were released from the glycoprotein by N-glycanase digestion, coupled to a 2-aminopyridyl residue, and subjected to two-dimensional sugar mapping technique. The major fractions consisted of "high-mannose type" oligosaccharides containing 8-9 mannose residues. Bi- and tri-antennary "complex type" oligosaccharides having terminal galactose were detected as minor components. These oligosaccharides were N-linked and contained no sialic acid. This structural profile is consistent with our previous characterization based on lectin-binding and glycosidase digestion. Functional specificity of identified chlamydial oligosaccharides was analyzed using glycopeptides fractionated from ovalbumin and structurally defined oligosaccharides from other sources. The glycopeptide fraction having high-mannose type oligosaccharide, as compared to those having complex or hybrid-type, showed a stronger inhibitory effect on attachment and infectivity of chlamydial organisms to HeLa cells. Among high-mannose type oligosaccharides, the strongest inhibition was observed with mannose 8 as compared with mannose 6, 7, or 9. These results indicate that a specific high-mannose type oligosaccharide linked to the major outer membrane protein of C. trachomatis mediates attachment and infectivity of the organism to HeLa cells.     

Occurrence of O-linked Xyl-GlcNAc and Xyl-Glc disaccharides in trocarin, a factor Xa homolog from snake venom

Summary.  Trocarin is a 46515-Da group D prothrombin-activating glycoprotein from the venom of the Australian elapid, Tropidechis carinatus . Amino acid sequencing and functional characterization of trocarin demonstrated that it is a structural and functional homolog of mammalian blood coagulation factor (F)Xa. In this study we show that, in contrast to mammalian Xa, which is not glycosylated, trocarin contains an O-linked carbohydrate moiety in its light chain and an N-linked carbohydrate oligosaccharide in its heavy chain. Mass spectrometry and sugar compositional analysis indicate that the O-linked carbohydrate moiety is a mixture of Xyl-GlcNAc-, GlcNAc-, Xyl-Glc- and Glc- structures linked to Ser 52. The N-linked carbohydrate on Asn 45 of the heavy chain is a sialylated, diantennary oligosaccharide that is located at the lip of the active site of the prothrombin activator.