Nono蛋白的原核表达、纯化和多克隆抗体的制备

目的表达和纯化带多聚组氨酸(6×His)标签的Nono(non-POU-domain-containing,octamer-bindingprotein)融合蛋白并制备抗Nono多克隆抗体。方法构建pET-28a( )-Nono重组表达质粒,转入Rosetta(DE3)大肠埃希菌,以IPTG诱导6×His-Nono融合蛋白表达,经镍离子金属螯合树脂纯化后,用纯化出的蛋白免疫BALB/C小鼠制备多克隆抗体,并用ELISA检测多克隆抗体的效价,Western印迹检测多克隆抗体的特异性。结果在大肠埃希菌中诱导出高水平表达的His-Nono融合蛋白,经亲和树脂纯化后免疫小鼠,获得了高特异性的抗Nono抗血清。结论成功构建pET-28a( )-Nono原核表达质粒,表达并纯化出高纯度的目标蛋白,制备出高滴度、高特异性的多克隆抗体。     

肺炎链球菌SpxA蛋白的原核表达、纯化及多克隆抗体的制备

目的 构建肺炎链球菌SpxA蛋白的原核表达系统,制备其多克隆抗体.方法 设计引物,利用PCR技术扩增肺炎链球菌D39菌株的spxA基因,并插入表达载体pET-28a(+)内,测序鉴定.重组质粒转化至大肠埃希菌BL21(DE3)中,以IPTG诱导表达含6个组氨酸标签的SpxA重组蛋白,经Ni-NTA亲和层析柱纯化后,以其为抗原免疫BALB/c小鼠制备多克隆抗体.用ELISA及Western印迹方法分别检测多克隆抗体的效价及特异性.结果 从大肠埃希菌中诱导出高表达的SpxA重组蛋白,纯化后免疫小鼠获得抗血清,ELISA测定其效价可达1:2 560 000以上,Western印迹结果显示其能特异性地作用于肺炎链球菌SpxA.结论 成功构建了pET-28a(+)-spxA原核表达质粒,获得了高纯度的目的 蛋白和高滴度、高特异性的多克隆抗体.     

程序死亡蛋白1基因的克隆及原核表达

目的 克隆人程序死亡蛋白(PD-1)基因并构建PD-1蛋白的原核表达质粒,在大肠埃希菌中进行表达.方法 用RT-PCR的方法从慢性乙肝患者外周血淋巴细胞总RNA中逆转录得到PD-1基因的cDNA,构建PD-1基因的原核表达质粒,在大肠埃希菌BL21(DE3)中进行表达并纯化.用SDS-PAGE、DNA测序、氨基酸测序等方法对表达蛋白进行鉴定.结果 克隆到PD-1基因编码区全长序列cDNA,经DNA测序与已报道的序列同源性达99.8%.构建得到PD-1的原核表达质粒,并在大肠埃希菌中表达,纯化获得纯度较高的PD-1蛋白.氨基酸测序证明表达蛋白的正确性.结论 成功克隆人PD-1基因,在大肠埃希菌中获得表达,得到纯化的PD-1蛋白,为进一步研究PD-1蛋白的功能及应用打下基础.     

人T-bet基因的表达、纯化及其免疫原性分析

目的：利用载体pQE30在大肠埃希菌（M15）原核表达人T-bet基因全长序列，并对表达产物进行纯化、免疫动物和制备多克隆抗体。方法：利用PCR技术从克隆载体pGEM-T／T-bet获得人T-bet的全长编码序列，并将其亚克隆至原核表达载体pQE30，形成重组表达质粒pT-bet；酶切鉴定挑选阳性重组质粒转化大肠埃希菌JM109，测序鉴定后转化M15，经IPTG370C诱导4h后，SDS-PAGE电泳、考马斯亮蓝染色判断以包涵体形式存在的带有6×His标签的融合蛋白（表达产物），用Ni^2＋ -IMAC层析柱对融合蛋白进行纯化；将纯化的蛋白免疫BALB／c小鼠，制备人T-bet的多克隆抗体。结果：成功表达和纯化了人T-bet，并制备了多克隆抗体，ELISA和Western blot结果显示了血清抗体的高效价（1：100000）和高度特异性。结论：成功构建了原核表达载体pT-bet，并在工程菌M15中获得大量表达，经纯化后获得高纯度的人T-bet蛋白，制备了效价和特异性良好的多克隆抗体，为进一步研究T-bet的生物学功能奠定了的基础。     

PBDC1蛋白的表达及其多克隆抗体的制备

目的原核表达、纯化PBDC1蛋白,制备PBDC1多克隆抗体。方法将PBDC1基因克隆到pET-28a(+)质粒中,构建成重组质粒pET-28a(+)-PBDC1。将此重组质粒转化大肠埃希菌BL21(DE3)后,IPTG诱导其在宿主菌中大量表达,并进行SDS-PAGE检测。经镍离子亲和柱纯化得到PBDC1融合蛋白,并以此免疫新西兰大白兔制备多克隆抗体。结果经质谱鉴定,获得了高纯度的PBDC1蛋白。经Western blot验证,获得了抗PBDC1蛋白的多克隆抗体。结论成功获得抗PBDC1蛋白的多克隆抗体,为研究PBDC1在红系分化中的功能提供了有利工具。     

牙龈卟啉菌外膜蛋白RagB的基因克隆、原核表达及其多克隆抗体的制备

目的:克隆牙龈卟啉菌外膜蛋白RagB基因、原核表达并制备牙龈卟啉菌外膜蛋白RagB多克隆抗体。方法:根据牙龈卟啉菌外膜蛋白RagB的DNA序列,设计特异性引物并经PCR扩增其基因。扩增产物经鉴定和测序分析后,与质粒pET-32a(+)重组形成pET-32a-ragB,继而转化大肠埃希菌。IPTG诱导重组蛋白的表达,再通过Ni-NTA亲和层析柱纯化后,用SDS-PAGE电泳和Western blot鉴定;获得的重组蛋白行常规家兔免疫,以制备多克隆抗体,并应用ELISA测定抗体效价。结果:PCR扩增获得编码区全长为1506bp可编码501个氨基酸的RagB基因,测序结果与GenBank公布的序列(AJ130872)完全一致;构建了pET-32a-ragB原核表达载体,获得了高表达的融合蛋白,SDS-PAGE电泳和Western blot表明具有较高的纯度;ELISA结果显示,特异性兔抗RagB的多克隆抗体效价为1×105。结论:在成功构建牙龈卟啉菌外膜蛋白RagB原核表达载体的基础上,高效表达及纯化了RagB融合蛋白,并制备了高效价的多克隆抗体。为进一步开展牙龈卟啉菌疫苗研究、建立牙龈卟啉菌感染的实验室诊断方法奠定了基础。     

中国旱獭β2m基因的克隆、原核表达及多克隆抗体的制备

目的 克隆、原核表达中国旱獭β2m基因,并制备多克隆抗体.方法 利用RT-PCR技术从中国旱獭脾细胞中扩增β2m基因,克隆至pET28a(+)质粒,构建原核表达载体pET-28a(+)-CWβ2m,再转化宿主菌Rosetta(DE3)pLacI诱导其表达.使用切胶回收纯化目的蛋白,将纯化蛋白免疫家兔制备多克隆抗体,并采用酶联免疫吸附实验检测抗体的灵敏度和特异性.结果 克隆出的中国旱獭β2m基因,与GenBank已公布的土拨鼠的碱基序列一致;Western印迹结果显示克隆的β2m基因能在大肠埃希菌中高效表达,免疫家兔获得了高效价的多克隆抗体.结论 成功克隆了中国旱獭β2m基因,在原核宿主中进行了高效表达,获得的多克隆抗体具有较高的效价.为人工制备MHC-Ⅰ类分子复合物,深入研究嗜肝病毒感染过程中特异性CTL应答和效应机制奠定了基础.     

TAT-EGFP融合蛋白的表达纯化及穿膜活性的研究

目的在大肠埃希菌中高效表达TAT-EGFP融合蛋白并鉴定纯化,然后进行穿膜活性的研究。方法以本室前期构建的重组质粒pQE-EGFP为模板,采用PCR方法特异性扩增TAT-EGFP基因序列,随后克隆于原核表达载体pQE-31,所构建的重组质粒经测序鉴定后转化大肠埃希菌JM109,IPTG诱导表达;SDS-PAGE和Western blotting鉴定表达蛋白质,经Ni2+-金属螯合亲和层析纯化,将纯化蛋白加入体外培养的小鼠黑色素瘤B16细胞中,荧光显微镜下观察TAT蛋白的穿膜活性。结果成功构建了pQE-TAT-EGFP重组质粒的,转化大肠埃希菌JM109,经IPTG诱导,目的蛋白表达率为25%,SDS-PAGE、Western blotting初步测定目的蛋白的相对分子质量(Mr)约为30160,纯化得到了目的融合蛋白TAT-EGFP。并在体外培养的B16细胞中证实TAT-EGFP融合蛋白穿透生物膜的能力。结论通过对TAT-EGFP融合蛋白穿膜活性的分析,证实了TAT具有穿膜活性,为TAT-PEⅢ融合蛋白抑瘤活性的研究提供了理论依据,也为生物大分子药物进入组织细胞内发挥治疗作用奠定了基础。     

单纯疱疹病毒Ⅰ和Ⅱ型共同抗原gD在大肠埃希菌中的重组表达

目的克隆人单纯疱疹病毒Ⅰ、Ⅱ(HSV-Ⅰ、Ⅱ)型共同性抗原gD基因,构建重组表达载体pMAL-c2/gD,诱导融合蛋白MBP-gD的表达.方法提取病毒DNA,PCR扩增出gD基因,克隆于原核表达载体pMAL-c2并转化大肠埃希菌DH5α.PCR、双酶切及测序证实插入的gD基因序列正确后,IPTG诱导表达融合蛋白MBP-gD,并进行免疫学鉴定.结果构建的重组表达质粒pMAL-c2/gD在大肠埃希菌中能高效表达.经SDS-PAGE分析,表达产物约占菌体总蛋白35.5%,其中39%以可溶蛋白形式存在于胞质中,61%以包涵体形式存在.结论构建了pMAL-c\+2/gD表达质粒,Western blot证实,HSV-Ⅰ gD单克隆抗体DL6可特异识别表达的gD蛋白,该蛋白具有天然gD的抗原性.     

重组原核质粒GST-hTudor-SN-SN(1～4)的构建及表达

目的 将人类Tudor-SN(tudor staphylococcal nuelease)蛋白SN(1～4)基因片段分别定向连入pGEX-4T-1质粒,使Tudor-SN蛋白sN各功能片段与(;"蛋白在大肠埃希菌BL21细胞内融合表达.方法 以重组质粒pSG5-Tudor-SN-flag为模板,PCR法扩增出目的 基因,利用EcoR Ⅰ和Sal Ⅰ双酶切法将目的 片段连接纠pGEX-4T-1载体卜,再将构建成功的GST-hTudor-SN-SN(1～4)重组质粒转化人大肠埃希菌BL-21内,IPTG诱导表达后再以考马斯亮蓝染色法检测GST融合蛋白的表达.结果 以单/双酶切和基因测序法鉴定构建的重组质粒均无误,考马斯亮监染色法观察到GST融合蛋白的正确表达.结论 重组原核GST.hTudor-SN-SN(1-4)质粒成功构建和表达.     

Klotho

The klotho gene was identified as an “aging-suppressor” gene in mice that accelerates aging when disrupted and extends life span when overexpressed. It encodes a single-pass transmembrane protein and is expressed primarily in renal tubules. The extracellular domain of Klotho protein is secreted into blood and urine by ectodomain shedding. The two forms of Klotho protein, membrane Klotho and secreted Klotho, exert distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors and functions as an obligate co-receptor for FGF23, a bone-derived hormone that induces phosphate excretion into urine. Mice lacking Klotho or FGF23 not only exhibit phosphate retention but also display a premature-aging syndrome, revealing an unexpected link between phosphate metabolism and aging. Secreted Klotho functions as a humoral factor that regulates activity of multiple glycoproteins on the cell surface, including ion channels and growth factor receptors such as insulin/insulin-like growth factor-1 receptors. Potential contribution of these multiple activities of Klotho protein to aging processes is discussed.     

Klotho: a fundamental regulator of aging

To escape aging and aging-related disorders has been one of mankind's biggest dreams from the beginning of history. However, our knowledge regarding the molecular mechanisms of aging has been limited. We recently developed a unique short lifespan mouse strain in which a single gene mutation caused multiple aging-related disorders and identified the responsible gene as klotho. The most characteristic phenotypes seem to be caused by abnormalities in calcium metabolism. Furthermore, the klotho gene is expressed principally in the important tissues for calcium homeostasis such as distal tubule cells of the kidney, choroid plexus in the brain, and the main cells of the parathyroid gland. Klotho plays a critical role for the regulation of calcium and phosphorus homeostasis by negatively regulating the synthesis of active Vitamin D. The deficiency of the klotho gene results in degradation of cells by the activation of calcium-dependent proteolysis in kidney, lung and heart. Importantly, the increased activation of calcium-dependent proteolysis occurs in the tissues of old mice together with the down regulation of klotho gene expression. What is Klotho protein required for and how does it act? In this review, I will discuss our working hypotheses on the biological roles and molecular functions of Klotho protein.     

Klotho基因及其与衰老相关的研究现状

在小鼠中Klotho基因的表达缺失导致出现了类似于人类衰老的综合征，本文综述了Klotho基因及蛋白的结构特点、Klotho表型异常的特性和Klotho基因的研究现状。     

Aging-suppressor Klotho: Prospects in diagnostics and therapeutics

IntroductionThe protein Klotho (KL) was first discovered in KL-deficient mice, which developed a syndrome similar to premature aging in humans. Since then, KL has been implicated in multiple molecular signaling pathways and diseases. KL has been shown to have anti-aging, healthspan and lifespan extending, cognitive enhancing, anti-oxidative, anti-inflammatory, and anti-tumor properties. KL levels decrease with age and in many diseases. Therefore, it has been of great interest to develop a KL-boosting or restoring drug, or to supplement endogenous Klotho with exogenous Klotho genetic material or recombinant Klotho protein, and to use KL levels in the body as a marker for the efficacy of such drugs and as a biomarker for the diagnosis and management of diseases.ObjectiveThe goal of this study was to provide a comprehensive review of KL levels across age groups in individuals who are healthy or have certain health conditions, using four sources: blood, cerebrospinal fluid, urine, and whole biopsy/necropsy tissue. By doing so, baseline KL levels can be identified across the lifespan, in the absence or presence of disease. In turn, these findings can be used to guide the development of future KL-based therapeutics and biomarkers, which will heavily rely on an individual’s baseline KL range to be efficacious.MethodsA total of 65 studies were collected primarily using the PubMed database. Research articles that were published up to April 2022 were included. Statistical analysis was conducted using RStudio.ResultsMean and median blood KL levels in healthy individuals, mean blood KL levels in individuals with renal conditions, and mean blood KL levels in individuals with metabolic or endocrine conditions were shown to decrease with age. Similarly, CSF KL levels in patients with AD also declined compared with age-matched controls.ConclusionsThe present study confirms the trend that KL levels in blood decrease with age in humans, among those who are healthy, and even further among those with renal and endocrine/metabolic illnesses. Further, by drawing this trend from multiple published works, we were able to provide a general idea of baseline KL ranges, specifically in blood in these populations. These data add to the current knowledge on normal KL levels in the body and how they change with time and in disease, and can potentially support efforts to create KL-based treatments and screening tools to better manage aging, renal, and metabolic/endocrine diseases.     

Klotho Deficiency Disrupts Hematopoietic Stem Cell Development and Erythropoiesis

Klotho deficiency is a characteristic feature of chronic kidney disease in which anemia and cardiovascular complications are prevalent. Disruption of the Klotho gene in mice results in hypervitaminosis D and a syndrome resembling accelerated aging that includes osteopenia and vascular calcifications. Given that the bone microenvironment and its cellular components considerably influence hematopoiesis, in the present study, we addressed the in vivo role of klotho in blood cell formation and differentiation. Herein, we report that genetic ablation of Klotho in mice results in a significant increase in erythropoiesis and a decrease in the hematopoietic stem cell pool size in the bone marrow, leading to impaired hematopoietic stem cell homing in vivo. Our data also suggest that high vitamin D levels are only partially responsible for these hematopoietic changes in Klotho^−/− mice. Importantly, we found similar hematopoietic abnormalities in Klotho^−/− fetal liver cells, suggesting that the effects of klotho in hematopoietic stem cell development are independent of the bone microenvironment. Finally, injection of klotho protein results in hematopoietic changes opposite to the ones observed in Klotho^−/− mice. These observations unveil a novel role for the antiaging hormone klotho in the regulation of prenatal and postnatal hematopoiesis and provide new insights for the development of therapeutic strategies targeting klotho to treat hematopoietic disorders associated with aging.Hematopoiesis is a complex and tightly regulated process of blood cell formation that is hierarchically coordinated. During normal hematopoiesis, diverse blood cell types are produced by the bone marrow (BM) in a manner related to physiologic requirement. Certain conditions may trigger additional production of blood cells. When the oxygen content of body tissues is low, the kidneys produce and release erythropoietin (Epo), a hormone that stimulates the BM to produce more red blood cells (RBCs). Aging is associated with disruption of normal hematopoiesis, resulting in an increase in the prevalence of anemia, the emergence of hematopoietic malignancies, and the development of leukemias.^1,2 Deterioration of vital organ function, such as kidney and heart, is also associated with age-related changes, as seen in chronic kidney disease (CKD) and cardiovascular disease (CVD).The antiaging hormone klotho, predominantly expressed in the kidneys, is emerging as a multifunctional protein regulating vital cellular functions.^3–5 Klotho was serendipitously discovered by Kuro-o et al⁶ when they observed symptoms of accelerated aging associated with a mutation in a specific gene in mice. Klotho exists in a membrane-bound form expressed at high levels in the kidney and, to a lesser extent, in other tissues, whereas a soluble form of klotho is secreted into blood, urine, and cerebrospinal fluid after cleavage of the extracellular domain.^7–10 Earlier studies convincingly demonstrate that membrane-bound klotho (α-klotho) is indispensable for signaling of the phosphatonin fibroblast growth factor 23 and that secreted klotho functions as an endocrine hormone responsible for the multiple organ defects observed in Klotho^−/− mice.^11–14Maintaining mineral ion homeostasis is critical and involves a delicate and concerted action between bone- and kidney-derived endocrine factors that operate through a complex feedback mechanism(s). Patients with CKD often present with bone diseases, such as osteopenia, osteoporosis, or osteomalacia, as a result of significant derangement of mineral metabolism.^15,16 In patients with CKD, failure of appropriate fibroblast growth factor 23/Klotho signaling results in hyperphosphatemia and vascular calcifications.¹⁷ Klotho expression is decreased progressively with loss of renal function,¹⁸ whereas blood levels of fibroblast growth factor 23 are elevated and are associated with increased CVD and mortality in these patients and in patients undergoing dialysis.^19–22 Moreover, abnormal blood cell production leading to severe anemia is a common complication in CKD and CVD and is caused by insufficient renal production of Epo.^23,24Disruption of the Klotho gene in mice due to mutations or inactivation (Klotho^−/− mice) results in growth retardation and early demise, osteopenia, extensive vascular calcifications, and skin atrophy, coupled with phosphate retention and hypervitaminosis D.^6,13,25–27 Conversely, overexpression of Klotho has been shown to rescue the klotho-deficient phenotype and extend the life span in mice, suggesting that Klotho functions as an aging suppressor gene in mammals.^6,28 Loss of klotho is further known to cause endothelial dysfunction by promoting oxidative stress.²⁹ It has been well appreciated that aging and oxidative stress adversely affect hematopoiesis by altering the niche functions.^30,31 An earlier report has also highlighted that klotho deficiency in mice results in reduced B lymphopoiesis, suggesting changes in immune regulatory functions by klotho.³² In addition, klotho expression at the mRNA level has been found to be significantly decreased in resting human CD4⁺ lymphocytes proportionally to advancing age.³³Signals emanating from the BM microenvironment and extrinsic soluble factors associated with the bone and marrow milieu are known to modulate hematopoietic stem cell (HSC) proliferation and differentiation.^34,35 Identifying the contributing factors involved in the regulation of hematopoiesis is an area of active research. Several lines of evidence highlight the role of bone-forming cells, the osteoblasts, in the HSC niche; postnatal depletion of osteoblasts negatively regulates the HSC pool size in the BM, whereas an increase in osteoblast number is associated with an augmentation in HSC number.^36–39 In addition, a series of advances indicate the importance of the bone-resorbing osteoclasts in regulation of the HSC microenvironment. Osteoclasts actively participate in HSC mobilization from the BM to the circulation and also promote formation of the HSC niche by controlling the maturation of osteoblasts.^40–44 Not only do bone cells participate in the regulation of hematopoiesis but the mineral content of the niche may also have a key function in localization of adult hematopoiesis, as reported in studies showing involvement of the calcium-sensing receptor and vitamin D signaling in this process.^45,46 Therefore, alterations in bone modeling and remodeling processes and/or mineralization seem to have a prominent effect on the modulation or formation of the hematopoietic niche. However, the regulation of mineral ion balance and hematopoiesis still remains largely a naive area.Because the bone environment and its components and the process of aging are closely linked to the regulation of hematopoiesis, and klotho deficiency is associated with a marked defect in skeletal mineralization and premature aging-like features, we hypothesized that klotho is involved in the regulation of RBC production and differentiation. In the present study, we demonstrate that loss of klotho severely affects erythropoiesis and HSC number and function. More important, we show that klotho affects hematopoiesis independently of changes in the BM environment and that the absence of klotho results in aberrant hematopoiesis prenatally, providing evidence for a novel and direct role for klotho in hematopoietic development. Although the kidney is the adult hematopoietic organ in zebra fish equivalent to mammalian BM,^47–49 the present data demonstrate for the first time, to our knowledge, a link between the kidney-bone-hematopoiesis axes in the mammalian system and attest that klotho is a key factor in the process of hematopoiesis.     

Klotho:与FPC蛋白相互作用的蛋白质分子

目的:利用酵母双杂交技术筛选与纤囊素相互作用的蛋白质,为进一步探讨纤囊素(FPC)在常染色体隐性遗传多囊肾病(ARPKD)发生、发展中的作用机制提供依据。方法:利用酵母双杂交系统以质粒pG-BKT7-FPC为"诱饵",在人类胚肾cDNA文库中筛选与FPC蛋白C末端相互作用宿主蛋白的基因,再通过一对一回交试验验证两者之间的相互作用。结果:酵母双杂交筛选得到相互作用的蛋白分子Klotho(后简称KL),回交试验再次确认KL能够与FPC蛋白相互作用。结论:FPC的C末端能够与KL相互作用,它们之间的相互作用可能为研究FPC在ARPKD发病中的功能及作用机制提供新途径。     

Nuclear localization of Klotho in brain: an anti-aging protein

Klotho is a putative age-suppressing gene whose overexpression in mice results in extension of life span. The Klotho gene encodes a single-pass transmembrane protein whose extracellular domain is shed and released into blood, urine, and cerebrospinal fluid, potentially functioning as a humoral factor. The extracellular domain of Klotho has an activity that increases the expression of antioxidant enzymes and confers resistance to oxidative stress in cultured cells and in whole animals. The transmembrane form of the Klotho protein directly binds to multiple fibroblast growth factor receptors and modifies their ligand affinity and specificity. The purpose of the present study was to determine the precise cellular localization of Klotho in the mouse brain. Using light microscopic immunohistochemical methods, we found the highest levels of Klotho immunoreactivity in 2 brain regions: the choroid plexus, and cerebellar Purkinje cells. In the choroid plexus cells, Klotho was found not only on the plasma membrane but also in large amounts near the nuclear membrane. Likewise, in the Purkinje cell Klotho was found throughout the cell including dendrites, axon and soma with large amounts near the nuclear membrane. Using immunoelectron microscopy, we found Klotho in the cell membrane, but the highest concentration was localized in the peripheral portion of the nucleus and the nucleolus in both cell types. This new finding suggests that in addition to Klotho being secreted from cells in brain, it also has a nuclear function.     

Involvement of the Klotho Protein in Dentin Formation and Mineralization

Klotho‐deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho‐deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho‐deficient mice–comparing the findings with those from their wild‐type littermates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein‐1 (DMP‐1), terminal deoxynucleotidyl transferase‐mediated deoxyuridinetriphosphate nick end‐labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho‐deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin‐predentinal border at the labial region. Several odontoblast‐like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP‐1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast‐like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast‐like cells and pulpal cells in the klotho‐deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho‐deficient dentin, except for the dentin around abnormal odontoblast‐like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization. Anat Rec, 2007. © 2008 Wiley‐Liss, Inc.     

Klotho: a novel regulator of calcium and phosphorus homeostasis

Klotho which was originally identified as an anti-aging protein is emerging as a substance with multiple effects on many systems including mineral homeostasis. In addition to its membrane-bound function as a co-receptor for fibroblast growth factor-23, soluble Klotho exerts effects as a circulating substance in plasma and urine. Novel features of this system include its autocrine–paracrine–endocrine glycan-modifying enzymatic function in the urinary lumen on calcium and phosphate transporters. Klotho induces phosphaturia by inhibiting the proximal tubule Na-coupled phosphate transporter. The action of Klotho is enzymatic in nature which includes alteration of transport activity and the more traditional means of regulation by trafficking. Klotho reduces calciuria by its distal as a sialidase directly on the apical calcium channel. Desialidation of the channel exposes glycan residues that promote binding to galectin-1, resulting in stabilization of residence on the plasma membrane. Through its systematic as well as renal actions, Klotho is emerging as a principal calciophosphoregulatory hormone.