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甲状旁腺激素(Parathyroid hormone/PTH)的定义

甲状旁腺激素（PTH）由甲状旁腺分泌，是一种含有84个氨基酸的多肽。

Parathyroid hormone (PTH) is secreted by the parathyroid glands as a polypeptide containing 84 amino acids.

甲状旁腺激素(Parathyroid hormone/PTH)的发现

1925年，科利普制备了甲状旁腺的热盐酸提取物，他正确地推断出这种方法需要从其他腺体基质成分中释放活性物质并使其可溶。作者表明，甲状旁腺的这些酸提取物可以完全缓解甲状旁腺切除术后的手足抽搐，并将甲状旁腺确立为分泌激素（PTH）的内分泌腺【1】。

In 1925, Collip prepared hot hydrochloric acid extracts of the parathyroid glands, an approach which he correctly deduced was needed to free the active substance from other gland stromal components and render it soluble. The author showed that these acid extracts of the parathyroid gland would completely relieve the tetany that followed parathyroidectomy, and established the parathyroids as an endocrine gland which secreted hormone (PTH) 【1】.

甲状旁腺激素(Parathyroid hormone/PTH)的结构特征

氨基酸组成：牛甲状旁腺激素已从脱脂甲状旁腺的三氯乙酸沉淀物中以均质形式分离出来。纯化激素的圆盘凝胶电泳显示单个电泳带，Edman氨基末端分析显示单个氨基酸PTH丙氨酸。纯化的激素由84个氨基酸组成。通过定时酸水解和总酶消化的组合确定的分离激素的氨基酸组成如下：Lys9，His4，Arg5，Trpl，Tyr1，Phe2，Leu8，ILe3，Met2，Val8，Ala7，Pro2，Gly4，Glu6，Gln5，Ser8，Asp6，Asn3。胱氨酸和苏氨酸不存在【2】。

Amino acid composition: Bovine parathyroid hormone has been isolated in homogeneous form from a trichloroacetic acid precipitate of defatted parathyroid glands. Disc gel electrophoresis of the purified hormone revealed a single electrophoretic band, and Edman amino terminal analysis showed a single amino acid, PTH-alanine. The purified hormone consisted of 84 amino acids.The amino acid composition of the isolated hormone, determined by a combination of timed acid hydrolysis and total enzymatic digestion, is as follows: Lys9, His4, Arg5, Trpl, Tyr1, Phe2, Leu8, ILe3, Met2, Val8, Ala7, Pro2, Gly4, Glu6, Gln5, Ser8, Asp6, Asn3. Cystine and threonine are absent【2】.

甲状旁腺激素(Parathyroid hormone/PTH)的作用机制

PTH作用于PTH/PTHrP受体（P1R），这是一种II类G蛋白偶联受体，可刺激腺苷酸环化酶/cAMP和磷脂酶C/肌醇磷酸（IP）信号通路。肽缺失研究表明，PTH的N末端残基在P1R激活中起着至关重要的作用【3】，交联和受体诱变研究表明，PTH的N末端残基与P1R中含有细胞外环的部分相互作用。跨膜螺旋的细胞外末端（近膜或J结构域）【4】。

PTH acts on the PTH/PTHrP receptor (P1R), a class II G protein-coupled receptor which stimulates the adenylyl cyclase/cAMP and phospholipase C/inositol phosphate (IP) signaling pathways. Peptide deletion studies have shown that the N-terminal residues of PTH play a crucial role in P1R activation 【3】 and cross-linking and receptor mutagenesis studies have revealed that the N-terminal residues of PTH interact with the portion of the P1R that contains the extracellular loops and extracellular ends of the transmembrane helices (the juxtamembrane or J domain) 【4】.

甲状旁腺激素(Parathyroid hormone/PTH)的类似物

甲状旁腺激素（PTH）-（1-14）和-（1-11）类似物受α-氨基异丁酸构象限制，通过PTH-1受体的近膜区域介导完全激动剂反应：甲状旁腺激素的N端部分对于PTH-1受体（P1R）激活至关重要，并且当与受体结合时被假定为α-螺旋。在一项研究中，研究了N端PTH寡肽中空间位阻和螺旋促进氨基酸α-氨基异丁酸（Aib）的取代是否会提高肽激活P1R的能力。分析[Ala3,12，Gln10，Har11，Trp14]PTH（1-14）NH2（[M]PTH（1-14））中每个位置的单个Aib取代对HKRK-B28细胞中cAMP刺激效力的影响显示Aib在大多数位置降低了效力；然而，位置1和3的Aib增强了效力。因此[Aib1,3，M]PTH（1-14）的效力比[M]PTH（1-14）高约100倍，比天然PTH（1-14）高约100000倍，比PTH（1-34）高2倍。较短的肽[Aib1,3，M]PTH（1-11）也完全有效，效力比[M]PTH（1-11）高1000倍。在表达P1R-delNt（一种缺乏大部分N端细胞外结构域的受体）的COS-7细胞中进行的cAMP刺激测定中，[Aib1,3，M]PTH（1-14）比[M]PTH（1-14）强50倍，比PTH（1-34）强1000倍。[Aib1,3，M]PTH（1-14），但不抑制PTH（1-34），抑制125I-[Aib1,3，Nle8，Gln10，Har11，Ala12，Trp14，Arg19，Tyr21]PTH（1-21）NH2与hP1R-delNt的结合。其他未修饰的PTH（1-34）中的Aib1,3取代增强了对hP1R-delNt的效力和结合亲和力，但它们对该肽对hP1R-WT没有影响。圆二色光谱表明Aib-1,3取代增加了所有测试肽的螺旋度，包括PTH（1-34）。因此，有人提出，PTH的N末端残基本质上是无序的，但在与PTH-1受体的激活结构域相互作用后，其构象受到限制，可能是a螺旋【5】。

Parathyroid Hormone (PTH)-(1-14) and -(1-11) Analogs Conformationally Constrained by a -Aminoisobutyric Acid Mediate Full Agonist Responses via the Juxtamembrane Region of the PTH-1 Receptor: The N-terminal portion of parathyroid hormone is critical for PTH-1 receptor (P1R) activation and has been postulated to be a-helical when bound to the receptor. In a study it was investigated whether substitution of the sterically hindered and helix-promoting amino acid a -aminoisobutyric acid (Aib) in N-terminal PTH oligopeptides would improve the capacity of the peptide to activate the P1R. Analysis of the effects of individual Aib substitutions at each position in [Ala3,12,Gln10,Har11,Trp14]PTH(1-14)NH2 ([M]PTH(1-14)) on cAMP-stimulating potency in HKRK-B28 cells revealed that Aib at most positions diminished potency; however, Aib at positions 1 and 3 enhanced potency. Thus [Aib1,3,M]PTH(1-14) was ~100-fold more potent than [M]PTH(1-14), ~100,000-fold more potent than native PTH(1-14), and 2-fold more potent than PTH(1-34). The shorter peptide, [Aib1,3,M]PTH(1-11), was also fully efficacious and 1,000-fold more potent than [M]PTH(1-11). In cAMP stimulation assays performed in COS-7 cells expressing P1R-delNt, a receptor that lacks most of the N-terminal extracellular domain, [Aib1,3,M]PTH(1-14) was 50-fold more potent than [M]PTH(1-14) and 1,000-fold more potent than PTH(1-34). [Aib1,3,M]PTH(1-14), but not PTH(1-34), inhibited the binding of 125I-[Aib1,3 ,Nle8 ,Gln10 ,Har11 ,Ala12 ,Trp14 ,Arg19 ,Tyr21]PTH(1-21)NH2 to hP1R-delNt. The Aib1,3 substitutions in otherwise unmodified PTH(1-34) enhanced potency and binding affinity on hP1R-delNt, but they had no effect for this peptide on hP1R-WT. Circular dichroism spectroscopy demonstrated that the Aib-1,3 substitutions increased helicity in all peptides tested, including PTH(1-34). Thus it has been suggested that the N-terminal residues of PTH are intrinsically disordered but become conformationally constrained, possibly as an a -helix, upon interaction with the activation domain of the PTH-1 receptor 【5】.

甲状旁腺激素(Parathyroid hormone/PTH)的功能

PTH及其片段及其类似物用于治疗骨质疏松症：骨质减少性骨骼创伤性骨折的易感性，以及由微结构恶化和骨丢失引起的正常身体运动引起的骨质疏松性骨骼自发性骨折，目前正在用抗吸收药物治疗，如双膦酸盐，降钙素，雌激素和选择性雌激素受体调节剂。这些抗吸收剂靶向破骨细胞，顾名思义，可以减少或阻止骨吸收。它们不能直接刺激骨形成，在卵巢切除的大鼠模型中增加骨量超过正常值，或改善微观结构。然而，有一系列药物-PTH及其一些片段及其类似物-可直接刺激骨骼生长并改善微观结构，而不损害破骨细胞。这些药物即将在临床上首次用于治疗骨质疏松症患者，并且可能在不久的将来用于加速骨折愈合。它们通过来自增殖失活的前成骨细胞，成骨细胞，骨细胞和骨衬里细胞上的1型PTH/PTH相关蛋白（PTHR1）受体的信号以三种方式刺激成骨细胞积聚和骨形成。受体信号关闭了前成骨细胞的增殖机制，并将其成熟为成骨细胞，导致成骨细胞产生并分泌几种因子，刺激没有PTHRI受体的骨祖细胞的广泛增殖，刺激骨衬里细胞向成骨细胞的逆转，并通过防止成骨细胞自杀性启动细胞凋亡来延长成骨细胞的寿命和生产力【6】。

PTH, its fragments and their analogs for the treatment of osteoporosis: The susceptibility to traumatic fracturing of osteopenic bones, and the spontaneous fracturing of osteoporotic bones by normal body movements caused by the microstructural deterioration and loss of bone, are currently treated with antiresorptive drugs, such as the bisphosphonates, calcitonin, estrogens, and selective estrogen receptor modulators. These antiresorptive agents target osteoclasts and, as their name indicates, reduce or stop bone resorption. They cannot directly stimulate bone formation, increase bone mass above normal values in ovariectomized rat models, or improve microstructure. However, there is a family of agents - the PTH and some of its fragments and their analogs - which directly stimulate bone growth and improve microstructure independently from impairing osteoclasts. These drugs are about to make their clinical debut in treating patients with osteoporosis and, probably not too far in the future, for accelerating fracture healing. They stimulate osteoblast accumulation and bone formation in three ways via signals from the type 1 PTH/PTH-related protein (PTHR1) receptors on proliferatively inactive preosteoblasts, osteoblasts, osteocytes and bone-lining cells. The receptor signals shut down the proliferative machinery in preosteoblasts and push their maturation to osteoblasts, cause the osteoblastic cells to make and secrete several factors that stimulate the extensive proliferation of osteoprogenitors without PTHRI receptors, stimulate the reversion of bone-lining cells to osteoblasts, and extend osteoblast lifespan and productivity by preventing them from suicidally initiating apoptosis 【6】.

甲状旁腺激素(Parathyroid hormone/PTH)的相关文献

1. Collip JB (1925). The extraction of a parathyroid hormone which will prevent or control parathyroid tetany and which regulates the level of blood calcium. Journal of Biological Chemistry, 63:395-438.
2. Brewer HB Jr, Ronan R (1970). Bovine Parathyroid Hormone: Amino Acid Sequence. Proceedings of the National Academy of Sciences, 67(4):1862-1869.
3. Takasu H, Gardella TJ, Luck MD, Potts JT, Jr and Bringhurst FR (1999). Amino-Terminal Modifications of Human Parathyroid Hormone (PTH) Selectively Alter Phospholipase C Signaling via the Type 1 PTH Receptor:  Implications for Design of Signal-Specific PTH Ligands. Biochemistry, 38:13453-13460. 
4. Hoare SRJ, Gardella TJ, and Usdin TB (2001). Evaluating the Signal Transduction Mechanism of the Parathyroid Hormone 1 Receptor. J. Biol. Chem., 276:7741-7753.
5. Shimizu N, Guo J, Gardella TJ (2001). Parathyroid Hormone (PTH)-(1-14) and -(1-11) Analogs Conformationally Constrained by a -Aminoisobutyric Acid Mediate Full Agonist Responses via the Juxtamembrane Region of the PTH-1 Receptor. J. Biol. Chem., 276 (52):49003-49012.
6. Whitfield JF, Morley P, Willick GE (2002). Parathyroid hormone, its fragments and their analogs for the treatment of osteoporosis. Treat Endocrinol., 1(3):175-190.

生物素可以与亲和素或者链霉亲和素有力结合，结合强度甚至接近共价键。生物素标记的肽通常用于免疫测定，组织细胞化学和基于荧光的流式细胞术。标记的抗生物素抗体也可以用来结合生物素化多肽。生物素标记常连接在赖氨酸侧链或者N末端。通常在多肽和生物素之间使用6-氨基己酸作为纽带，纽带能够灵活结合底物，并且在有空间位阻的情况下能结合地更好。专肽生物根据需求，向客户提供具有不同位点生物素标记多肽的定制合成。

专肽生物合成用于蛋白质-蛋白质相互作用研究的生物素化肽。尽管生物素可以在 N 端或 C 端引入（通过赖氨酸残基），但我们建议使用 N 端修饰，因为它成本低、成功率高、周转时间短且易于操作。因为多肽合成是从 C 端到 N 端合成的，因此，N 端修饰是 SPPS步骤的最后一步，不需要额外的特定缩合步骤。相比之下，C 端修饰需要额外的步骤，并且通常更复杂。当然，原则上生物素可以定位在任何地方。

生物素可以通过多种不同的接头或间隔物与肽分离。尽管如此，还是建议包含一个灵活的间隔物，例如 Ahx（一个 6 碳接头），以使生物素标签更加稳定或灵活。

专肽生物在 N 端或 C 端提供生物素化：生物素-N 端、赖氨酸-生物素-肽中间和赖氨酸-生物素-C 端。
专肽生物还可以使用 Ahx 接头或长碳 (LC) 接头提供生物素化：生物素-Ahx-N 末端、Lys-Ahx-生物素-肽中间、Lys-Ahx-生物素-C-末端。

（生物素结构）

示例：
GRGDS在N端和C端标记生物素的结构展示。

1、GRGDS在N端标记生物素，不增加Ahx 接头

2、GRGDS在N端标记生物素，增加一个Ahx 接头

3、GRGDS在C端标记生物素，不增加Ahx 接头

4、GRGDS在C端标记生物素，增加一个Ahx 接头。