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Isolation of Keratan Sulfate Disaccharide-branched Chondroitin Sulfate E from Mactra chinensis
中国蛤蜊中硫酸角质素二糖-支链硫酸软骨素E的分离   

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参见作者原研究论文

本实验方案简略版
Biochemical Journal
Sep 2016

Abstract

Glycosaminoglycans (GAGs) including chondroitin sulfate (CS), dermatan sulfate (DS), heparin (HP), heparan sulfate (HS) and keratan sulfate (KS) are linear, sulfated repeating disaccharide sequences containing hexosamine and uronic acid (or galactose in the case of KS). Recently, a keratan sulfate (KS) disaccharide [GlcNAc6S(β1-3)Galactose(β1-]-branched CS-E was identified from the clam species M. chinensis. Here, we report the isolation protocol for KS-branched CS from M. chinensis.

Keywords: Mactra chinensis (中国蛤蜊), Glycosaminoglycan (糖胺聚糖), Chondroitin sulfate (硫酸软骨素), Keratan sulfate (硫酸角质素), Galactose (半乳糖)

Background

GAGs are found in tissues as the glycan moieties of proteoglycan (PG) glycoconjugates. CS is a GAG type composed of linear, sulfated repeating disaccharide sequences of N-acetyl-D-galactosamine (GalNAc) and glucuronic acid (GlcA). Another GAG type, DS, is biosynthesized through the action of glucuronyl C5-epimerase on CS, converting its GlcA to the CS epimer, iduronic acid (IdoA). The other GAG types HS and HEP are consisted of sulfated repeating disaccharide sequence of N-acetyl-D-glucosamine (GlcNAc) and GlcA/IdoA. KS is composed of sulfated repeating disaccharide sequences of GlcNAc and galactose. Among them, structural variations of CS, such as sulfation patterns and fucosylation, depend on the species and tissue of origin. For example, the A-unit with the structure [-4)GlcA(β1-3)GalNAc4S(β1-] (where S designates a sulfonate residue) is a predominant disaccharide found in mammalian or chicken tracheal cartilage CS, while the C-unit with the structure [-4)GlcA(β1-3)GalNAc6S(β1-] is a major disaccharide found in shark cartilage or salmon nasal cartilage CS. In contrast, a significant amount of the D-unit disaccharide [-4)GlcA2S(β1-3)GalNAc6S(β1-] is characteristically found in CS isolated from shark cartilage, while the E-unit disaccharide [-4)GlcA(β1-3)GalNAc4S,6S(β1-] is characteristic in squid cartilage. In sea cucumber, a sulfated fucose branches at the 3-OH position of GlcA. The disaccharide composition of CS governs its biological activities, including cell proliferation, migration, differentiation, cell-cell crosstalk, adhesion and wound repair through the interaction with growth factors, receptors, and other CS-binding proteins. Evidence suggests that CS structure is tightly correlated with function. For example, consecutive and disulfated disaccharide units including B, D and E-units in CS are critical for the interaction between CS and binding proteins (Hikino et al., 2003). A branched fucose at the 3-OH position of GlcA is also required for the anticoagulant activity of fucosylated CS (Mourão et al., 1996).

In general, isolation of GAGs is carried out as follows. 1) acetone defatting, 2) proteolysis, 3) collection of the GAGs, 4) fractionation of GAGs by anion-exchange chromatography and 5) desalting (Maccari et al., 2015). In our protocol, actinase E from Streptomyces griseus (step 2) and cetylpyridinium chloride precipitation (step 3) were used for the isolation of GAGs.

Materials and Reagents

  1. 200 and 1,000 μl pipette tips (Thermo Fisher Scientific)
  2. Centrifuge tube
  3. Spectra/Por®7 Dialysis Membrane Pre-treated RC Tubing MWCO: 3,500 (Spectrum, catalog number: 132111 )
  4. HiPrepTM DEAE FF 16/10 (GE Healthcare, catalog number: 28936541 )
  5. Dry powder of hot water extract from M. chinensis viscera obtained from Futtsu City Fishery Association in Chiba, Japan
  6. Acetone (Wako Pure Chemical Industries, catalog number: 011-00357 )
  7. Acetic acid (NACALAI TESQUE, catalog number: 00212-43 )
  8. Perchloric acid (60%, w/v) (NACALAI TESQUE, catalog number: 26502-85 )
  9. Ethanol (99.5% w/v) (NACALAI TESQUE, catalog number: 14713-53 )
  10. Tris (hydroxymethyl)aminomethane (Tris) (NACALAI TESQUE, catalog number: 35406-91 )
  11. Actinase E (Funakoshi, catalog number: KA-001 )
  12. Sodium hydroxide (NaOH) (NACALAI TESQUE, catalog number: 31511-05 )
  13. Sodium tetrahydroborate (Wako Pure Chemical Industries, catalog number: 192-01472 )
  14. Cetylpyridinium chloride monohydrate (99.0-102.0% w/v) (Wako Pure Chemical Industries, catalog number: 086-06683 )
  15. Hydrochloric acid (HCl) (35.0% w/v) (NACALAI TESQUE, catalog number: 18321-05 )
  16. Sodium chloride (NaCl) (NACALAI TESQUE, catalog number: 31320-05 )
  17. Sodium dihydrogenphosphate, anhydrous (NaH2PO4) (NACALAI TESQUE, catalog number: 31720-65 )
  18. Di-sodium hydrogenphosphate (Na2HPO4) (NACALAI TESQUE, catalog number: 31801-05 )
  19. Tris acetate buffer (pH 8.0) (see Recipes)
  20. NaOH buffer (see Recipes)
  21. Cetylpyridinium chloride solution (see Recipes)
  22. 50 mM sodium phosphate (pH 6) (see Recipes)
  23. 2.0 M NaCl in 50 mM sodium phosphate (pH 6) (see Recipes)

Equipment

  1. Refrigerated centrifuge (Sakuma, model: M200-IVD )
  2. Fume hood
  3. Erlenmeyer flask (AGC, catalog number: 4980FK500 )
  4. Stainless steel spoon (180 mm)
  5. Pipettes (Gilson, models: P20 , P200 and P1000 )
  6. Corning® reusable low form beaker, polypropylene, size 3 L (Corning, catalog number: 1000P-3L )
  7. Stir bar
  8. BioShaker (TAITEC, model: BR-23FP MR )
  9. Freeze drier (TOKYO RIKAKIKAI, Eyela, model: FDU-830 )
  10. Gradient pump (Bio-Rad Laboratories, model: ECONO GRADIENT PUMP )
  11. Analytical balance (Shimadzu, model: ATX224 )

Procedure

  1. Isolation of crude GAGs
    1. To remove lipids from the sample, suspend the dried powder (5 g) in a centrifuge tube with 20 ml of acetone and then centrifuge sample at 2,300 x g for 15 min at room temperature. Remove supernatant. Repeat this defatting step three times. After centrifugation, dry samples overnight in fume hood at room temperature. In general, 30 g of dried powder was defatted.
    2. To digest the core proteins, dissolve the defatted sample in 120 ml of Tris acetate buffer (pH 8.0) (see Recipes) containing actinase E in an Erlenmeyer flask and maintain for 18 h at 45 °C with shaking at 100 rpm.
    3. Add 600 ml of NaOH buffer (see Recipes) to the sample and leave to stand at 4 °C for 18 h.
    4. Neutralize the sample with 100 ml of 2.0 N acetic acid at room temperature and precipitate the degraded proteins by adding perchloric acid (to a final concentration of 5%). Separate the degraded proteins by centrifugation at 2,300 x g for 15 min at 4 °C.
    5. Transfer the supernatant into a dialysis membrane (MWCO: 3,500) and dialyze it against distilled water at room temperature for 16 h.
    6. Transfer the dialyzed sample solution into a new Erlenmeyer flask. Add CPC solution (see Recipes) (final concentration 0.1%) and suspend well. Cetylpyridinium chloride (CPC), a cationic surfactant, interacts with the sulfates and carboxylates of GAGs. After storage at 4 °C for 16 h, GAG-CPC complex in the Erlenmeyer flask can be observed.
    7. Collect the GAG-CPC complex by centrifugation at 10,000 x g for 15 min at 4 °C and discard the supernatant.
    8. Transfer the precipitate (GAG-CPC complex) into a new conical tube using a stainless steel spoon (180 mm). To remove CPC from GAG-CPC complex, resuspend the precipitate in 4 ml of 2.5 M NaCl using a 1,000 μl pipette. Centrifuge the solution at 10,000 x g for 15 min at 4 °C and transfer the GAG-containing supernatant into a new conical tube.
    9. Suspend with 11 volumes of 85% ethanol and store at 4 °C for 16 h. After incubation, collect crude GAGs by centrifugation at 10,000 x g for 15 min at 4 °C and remove the supernatant. Dissolve the precipitate with 10 ml of distilled water.
    10. Transfer the supernatant fluid (dissolved precipitate) into a dialysis membrane (MWCO: 3,500) and dialyze it against 2 L of distilled water in a polypropylene beaker (3 L) with a stir bar at room temperature for 16 h to remove excess NaCl from crude GAGs. Distilled water is changed 5 times during dialysis.
    11. Transfer the dialyzed sample into a new conical tube and freeze.
    12. Lyophilize overnight using freeze drier to obtain the crude GAG powder.

  2. Fractionation of KS-branched CS-E
    1. Dissolve the crude GAGs (30 mg) into 2 ml of distilled water.
    2. The dissolved crude GAGs are fractionated using low-pressure liquid chromatography. Fractionation condition: ECONO GRADIENT PUMP (Bio-Rad Laboratories) is applied at a flow rate of 2 ml/min on a HiPrep DEAE FF at room temperature. The eluent buffers are as follows: (A) 50 mM sodium phosphate (pH 6) (see Recipes), (B) 2.0 M NaCl in 50 mM sodium phosphate (pH 6) (see Recipes). The gradient program is 0-30 min (5% B), 30-150min (5-100% B), and 150-180 min (100% B).
    3. Collect fractionated samples at 30 min intervals. Dry the fractionated samples using a freeze drier and keep samples at 4 °C.

Data analysis

The dried powder (crude GAGs or fractionated CS) after freeze-drying was weighed using analytical balance.

Notes

  1. Dry powder of hot water extracted viscera from M. chinensis was used.
  2. Crude GAGs contain heparan sulfate and chondroitin sulfate.
  3. 353.8 mg of crude GAG/g of dry powder was recovered from the viscera of M. chinensis (Higashi et al., 2016).
  4. CS usually elutes at 90 to 150 min when fractionation is performed by anion-exchange chromatography, whereas elution time of hyaluronic acid (HA) and HS is faster than that of CS. HA eluted at 0 to 30 min, and HS mainly eluted at 60 to 90 min.
  5. In case of M. chinensis, fractionated CS was obtained in fraction 3 (18.5 mg) and in Fraction 4 (3.73 mg) from 30 mg of crude GAGs (Higashi et al., 2016).

Recipes

  1. Tris acetate buffer (pH 8.0)
    50 mM Tris acetate (pH 8.0)
    2.5 mg/ml of actinase E
  2. NaOH buffer
    0.5 N NaOH
    0.3 M sodium tetrahydroborate
  3. Cetylpyridinium chloride solution
    5% hexadecylpyridinium (Cetylpyridinium) chloride monohydride
    30 mM NaCl
  4. 50 mM sodium phosphate (pH 6)
    50 mM sodium dihydrogenphosphate, anhydrous (NaH2PO4)
    50 mM di-sodium hydrogenphosphate (Na2HPO4)
  5. 2.0 M NaCl in 50 mM sodium phosphate (pH 6)
    50 mM sodium dihydrogenphosphate, anhydrous (NaH2PO4)
    50 mM di-sodium hydrogenphosphate (Na2HPO4)
    2.0 M NaCl

Acknowledgments

We thank Dr. Sayaka Masuko for her help in preparing this manuscript. This protocol was adopted from the original work (Higashi et al., 2016) to provide the detailed procedures. This study was supported in part by the Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sport, Science and Technology of Japan and the Inohana Foundation, Chiba University.

References

  1. Higashi, K., Takeda, K., Mukuno, A., Okamoto, Y., Masuko, S., Linhardt, R. J. and Toida, T. (2016). Identification of keratan sulfate disaccharide at C-3 position of glucuronate of chondroitin sulfate from Mactra chinensis. Biochem J 473(22): 4145-4158.
  2. Hikino M, Mikami T, Faissner A, Vilela-Silva AC, Pavão MS, Sugahara K. Oversulfated dermatan sulfate exhibits neurite outgrowth-promoting activity toward embryonic mouse hippocampal neurons: implications of dermatan sulfate in neuritogenesis in the brain. (2003). J Biol Chem 278(44):43744-54.
  3. Maccari, F., Galeotti, F., Volpi, N. (2015). Isolation and structural characterization of chondroitin sulfate from bony fishes. Carbohydr Polym 129:143-147.
  4. Mourão PA, Pereira MS, Pavão MS, Mulloy B, Tollefsen DM, Mowinckel MC, Abildgaard U. (1996). Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. Sulfated fucose branches on the polysaccharide account for its high anticoagulant action. J Biol Chem 271(39):23973-84.

简介

包括硫酸软骨素(CS),硫酸皮肤素(DS),肝素(HP),硫酸乙酰肝素(HS)和硫酸角质素(KS)的糖胺聚糖(GAGs)是含有己糖胺和糖醛酸(或半乳糖 KS案)。 最近,从蛤蜊种群中鉴定了硫酸角质素(KS)二糖[GlcNAc6S(β1-3)半乳糖(β1 - ] - 支链的CS-E),这里我们报告了分离方案 用于来自中华猕猴桃的KS分枝CS。
【背景】在组织中发现GAG作为蛋白多糖(PG)糖缀合物的聚糖部分。 CS是由N,N-乙酰基-D-半乳糖胺(GalNAc)和葡萄糖醛酸(GlcA)的线性,硫酸化重复二糖序列组成的GAG型。另一种GAG类型的DS通过葡萄糖醛酸C5-差向异构酶对CS的作用进行生物合成,将其GlcA转化为CS差向异构体,艾杜糖醛酸(IdoA)。其他GAG类型HS和HEP由N,N-乙酰基-D-氨基葡糖(GlcNAc)和GlcA / IdoA的硫酸化重复二糖序列组成。 KS由GlcNAc和半乳糖的硫酸化重复二糖序列组成。其中,CS的结构变化,如硫酸化模式和岩藻糖基化,取决于物种和组织的来源。例如,具有结构[-4] GlcA(β1-3)GalNAc4S(β1-)(其中S表示磺酸盐残基)的A-单元是在哺乳动物或鸡气管软骨CS中发现的主要二糖,而C-具有结构[-4] GlcA(β1-3)的单元GalNAc6S(β1-)是在鲨鱼软骨或鲑鱼鼻软骨CS中发现的主要二糖,相比之下,D单位二糖[-4] GlcA2S (β1-3)GalNAc6S(β1-)特征在于从鲨鱼软骨分离的CS中发现,而E单位二糖[-4] GlcA(β1-3)GalNAc4S,6S(β1-)在鱿鱼软骨中是特征性的海参,GlcA 3-OH位置的硫酸岩藻分枝,CS的二糖组成通过与生长因子的相互作用控制其生物活性,包括细胞增殖,迁移,分化,细胞间串扰,粘附和伤口修复,受体和其他CS结合蛋白。证据表明CS结构紧密相关功能。例如,CS中包括B,D和E单元的连续和二硫酸化二糖单元对于CS和结合蛋白之间的相互作用是至关重要的(Hikino等人,2003)。 GlcA的3-OH位置处的支链岩藻糖也是岩藻糖化CS的抗凝血活性所必需的(Mourãoet al。,1996)。
一般来说,GAG的隔离如下进行。 1)丙酮脱脂,2)蛋白水解,3)GAG的收集,4)通过阴离子交换色谱法分离GAG,5)脱盐(Maccari等人,2015)。在我们的方案中,来自灰色链霉菌(Streptomyces griseus)(步骤2)的肌动蛋白酶E和氯化十六烷基吡啶鎓沉淀(步骤3)用于分离GAG。

关键字:中国蛤蜊, 糖胺聚糖, 硫酸软骨素, 硫酸角质素, 半乳糖

材料和试剂

  1. 200和1,000μl移液器吸头(Thermo Fisher Scientific)
  2. 离心管
  3. Spectra / Por ®透析膜预处理RC管材MWCO:3,500(光谱,目录号:132111)
  4. HiPrep TM DEAE FF 16/10(GE Healthcare,目录号:28936541)
  5. 来自M的热水提取物的干粉。日本千叶市富津市渔业协会获得的中华鲟内脏
  6. 丙酮(和光纯药工业公司,目录号:011-00357)
  7. 乙酸(NACALAI TESQUE,目录号:00212-43)
  8. 高氯酸(60%,w / v)(NACALAI TESQUE,目录号:26502-85)
  9. 乙醇(99.5%w / v)(NACALAI TESQUE,目录号:14713-53)
  10. 三羟甲基氨基甲烷(Tris)(NACALAI TESQUE,目录号:35406-91)
  11. 肌动蛋白酶E(Funakoshi,目录号:KA-001)
  12. 氢氧化钠(NaOH)(NACALAI TESQUE,目录号:31511-05)
  13. 四氢硼酸钠(和光纯药,目录号:192-01472)
  14. 氯化十六烷基吡啶鎓一水合物(99.0-102.0%w / v)(Wako Pure Chemical Industries,目录号:086-06683)
  15. 盐酸(HCl)(35.0%w / v)(NACALAI TESQUE,目录号:18321-05)
  16. 氯化钠(NaCl)(NACALAI TESQUE,目录号:31320-05)
  17. 无水磷酸二氢钠(NaH 2 PO 4)(NACALAI TESQUE,目录号:31720-65)
  18. 磷酸氢二钠(Na 2 HPO 4)(NACALAI TESQUE,目录号:31801-05)
  19. Tris醋酸缓冲液(pH 8.0)(参见食谱)
  20. NaOH缓冲液(参见食谱)
  21. 十六烷基氯化吡啶溶液(参见食谱)
  22. 50mM磷酸钠(pH6)(参见食谱)
  23. 在50mM磷酸钠(pH6)中的2.0M NaCl(参见食谱)

设备

  1. 冷冻离心机(Sakuma,型号:M200-IVD)
  2. 通风柜
  3. 锥形瓶(AGC,目录号:4980FK500)
  4. 不锈钢勺子(180毫米)
  5. 移液器(Gilson,型号:P20,P200和P1000)
  6. Corning ®可重复使用的低烧杯,聚丙烯,尺寸3 L(康宁,目录号:1000P-3L)
  7. 搅拌棒
  8. BioShaker(TAITEC,型号:BR-23FP MR)
  9. 冷冻干燥机(TOKYO RIKAKIKAI,Eyela,型号:FDU-830)
  10. 梯度泵(Bio-Rad Laboratories,型号:ECONO GRADIENT PUMP)
  11. 分析天平(Shimadzu,型号:ATX224)

程序

  1. 粗GAG的分离
    1. 为了从样品中除去脂质,将干燥的粉末(5g)用20ml丙酮悬浮在离心管中,然后在室温下以2,300×g离心样品15分钟。去除上清液。重复这个脱脂步骤三次。离心后,室温干燥样品通风柜过夜。一般来说,将30g干粉脱脂
    2. 为了消化核心蛋白质,将脱脂样品溶解在含有肌动蛋白酶E的120ml Tris乙酸盐缓冲液(pH8.0)(参见食谱)中,在锥形瓶中,并在45℃下以100rpm振荡保持18小时。 >
    3. 向样品中加入600毫升的NaOH缓冲液(参见食谱),并在4℃静置18小时
    4. 在室温下用100ml的2.0N乙酸中和样品,并通过加入高氯酸(最终浓度为5%)沉淀出降解的蛋白质。通过在4℃下以2,300×g离心分离降解的蛋白质15分钟。
    5. 将上清液转移到透析膜(MWCO:3,500)中,并在室温下用蒸馏水透析16小时。
    6. 将透析的样品溶液转移到新的锥形瓶中。添加CPC解决方案(参见配方)(终浓度为0.1%)并暂停。氯化十六烷基吡啶(CPC),阳离子表面活性剂与GAG的硫酸盐和羧酸盐相互作用。在4℃下保存16小时后,可以观察到锥形瓶中的GAG-CPC复合物。
    7. 通过在4℃下以10,000×g离心15分钟收集GAG-CPC复合物,并弃去上清液。
    8. 使用不锈钢匙(180毫米)将沉淀物(GAG-CPC复合物)转移到新的锥形管中。为了从GAG-CPC复合物中除去CPC,使用1,000μl移液管将沉淀物重新悬浮在4 ml的2.5 M NaCl中。在4℃下以10,000xg离心溶液15分钟,并将含有GAG的上清液转移到新的锥形管中。
    9. 用11体积的85%乙醇悬浮并在4℃下储存16小时。孵育后,通过在4℃以10,000×g离心15分钟收集粗GAG,并除去上清液。用10ml蒸馏水溶解沉淀物
    10. 将上清液(溶解的沉淀物)转移到透析膜(MWCO 3,500)中,并在聚丙烯烧杯(3升)中用搅拌棒在2小时的蒸馏水中在室温下透析16小时以从粗产物中除去过量的NaCl聚糖。蒸馏水在透析期间改变5次。
    11. 将透析的样品转移到新的锥形管中并冻结。
    12. 使用冷冻干燥器将其冻干过夜,得到粗GAG粉末
  2. KS分支CS-E的分馏
    1. 将粗GAG(30mg)溶解于2ml蒸馏水中
    2. 使用低压液相色谱分离溶解的粗GAG。分级条件:在室温下,在HiPrep DEAE FF上以2ml / min的流速施用ECONO GRADIENT PUMP(Bio-Rad Laboratories)。洗脱液缓冲液如下:(A)50mM磷酸钠(pH6)(参见食谱),(B)在50mM磷酸钠(pH6)中的2.0M NaCl(参见食谱)。梯度程序为0-30分钟(5%B),30-150分钟(5-100%B)和150-180分钟(100%B)。
    3. 以30分钟间隔收集分级样品。使用冷冻干燥器干燥分馏的样品,并将样品保持在4°C

数据分析

使用分析天平称量冷冻干燥后的干燥粉末(粗GAG或分馏CS)。

笔记

  1. 使用热水中的干粉从中华鲟提取的内脏
  2. 粗GAG含有硫酸乙酰肝素和硫酸软骨素
  3. 从M的内脏回收353.8mg粗GAG / g干粉。 chinensis (Higashi等人,2016)。
  4. 当通过阴离子交换层析分离时,CS通常在90至150分钟洗脱,而透明质酸(HA)和HS的洗脱时间比CS快。 HA在0至30分钟内洗脱,HS主要在60至90分钟时洗脱
  5. 在M的情况下。中成药,从30mg粗GAG(Higashi等人,2016)获得级分3(18.5mg)和级分4(3.73mg)中的分级CS。

食谱

  1. Tris醋酸缓冲液(pH 8.0)
    50mM Tris乙酸盐(pH 8.0)
    2.5 mg / ml肌动蛋白酶E
  2. NaOH缓冲液
    0.5 N NaOH
    0.3M四氢硼酸钠
  3. 氯化十六烷基吡啶溶液
    5%十六烷基吡啶鎓(Cetylpyridinium)氯化物一水合物
    30 mM NaCl
  4. 50mM磷酸钠(pH6)
    50mM磷酸二氢钠,无水(NaH 2 PO 4·4)
    50mM磷酸氢二钠(Na 2 HPO 4)
  5. 在50mM磷酸钠(pH6)中的2.0M NaCl
    50mM磷酸二氢钠,无水(NaH 2 PO 4·4)
    50mM磷酸氢二钠(Na 2 HPO 4)
    2.0 M NaCl

致谢

我们感谢Sayaka Masuko博士帮助准备这份手稿。该协议是从原始作品(Higashi等人,2016)中获得的,以提供详细的程序。这项研究部分得到了日本教育,文化,体育,科技和科技研究部门的支持,日本千叶大学的Inohana基金会。

参考

  1. Higashi,K.,Takeda,K.,Mukuno,A.,Okamoto,Y.,Masuko,S.,Linhardt,RJ和Toida,T。(2016)。来自中华猕猴桃的硫酸软骨素葡萄糖醛酸C-3位的硫酸角质素二糖鉴定 。生物化学J 473(22):4145-4158。
  2. Hikino M,Mikami T,Faissner A,Vilela-Silva AC,PavãoMS,Sugahara K.  硫酸化硫酸皮肤素对胚胎小鼠海马神经元表现出神经突促生长活性:硫酸皮肤素对大脑神经发生的影响。(2003)。 J Biol Chem 278(44):43744-54。
  3. Maccari,F.,Galeotti,F.,Volpi,N。(2015)。来自骨骼鱼的硫酸软骨素的分离和结构表征 Carbohydr Polym 129:143-147。
  4. MourãoPA,Pereira MS,PavãoMS,Mulloy B,Tollefsen DM,Mowinckel MC,Abildgaard U.(1996)。棘皮动物中岩藻糖化硫酸软骨素的结构和抗凝活性。多糖上的硫酸化岩藻糖分支具有高抗凝作用。生物化学 271(39):23973-84。
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引用:Higashi, K. and Toida, T. (2017). Isolation of Keratan Sulfate Disaccharide-branched Chondroitin Sulfate E from Mactra chinensis. Bio-protocol 7(15): e2441. DOI: 10.21769/BioProtoc.2441.
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