Identification of membrane-bound serine proteinase matriptase as processing enzyme of insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1/angiomodulin/mac25)

Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) modulates cellular adhesion and growth in an IGF/insulin-dependent or independent manner. It also shows tumor-suppressive activity in vivo. We recently found that a single-chain IGFB-rP1 is proteolytically cleaved to a two-chain form by a trypsin-like, endogenous serine proteinase, changing its biological activities. In this study, we attempted to identify the IGFBP-rP1-processing enzyme. Of nine human cell lines tested, seven cell lines secreted IGFBP-rP1 at high levels, and two of them, ovarian clear cell adenocarcinoma (OVISE) and gastric carcinoma (MKN-45), highly produced the cleaved IGFBP-rP1. Serine proteinase inhibitors effectively blocked the IGFBP-rP1 cleavage in the OVISE cell culture. The conditioned medium of OVISE cells did not cleave purified IGFBP-rP1, but their membrane fraction had an IGFBP-rP1-cleaving activity. The membrane fraction contained an 80-kDa gelatinolytic enzyme, which was identified as the membrane-type serine proteinase matriptase (MT-SP1) by immunoblotting. When the membrane fraction was separated by SDS/PAGE, the IGFBP-rP1-cleaving activity comigrated with matriptase. A soluble form of matriptase purified in an inhibitor-free form efficiently cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved IGFBP-rP1. Furthermore, small interfering RNAs for matriptase efficiently blocked both the matriptase expression and the cleavage of IGBP-rP1 in OVISE cells. These results demonstrate that IGFBP-rP1 is processed to the two-chain form by matriptase on the cell surface.     

The cell surface receptor FGFRL1 forms constitutive dimers that promote cell adhesion

FGFRL1 is a novel member of the fibroblast growth factor (FGF) receptor family. Utilizing the FRET (fluorescence resonance energy transfer) technique, we demonstrate that FGFRL1 forms constitutive homodimers at cell surfaces. The formation of homodimers was verified by co-precipitation of differentially tagged FGFRL1 polypeptides from solution. If overexpressed in cultivated cells, FGFRL1 was found to be enriched at cell-cell contact sites. The extracellular domain of recombinant FGFRL1 promoted cell adhesion, but not cell spreading, when coated on plastic surfaces. Adhesion was mediated by heparan sulfate glycosaminoglycans located at the cell surface. It could specifically be blocked by addition of soluble heparin but not by addition of other glycosaminoglycans. When the amino acid sequence of the putative heparin-binding site was modified by in vitro mutagenesis, the resulting protein exhibited decreased affinity for heparin and reduced activity in the cell-binding assay. Moreover, a synthetic peptide corresponding to the heparin-binding site was able to neutralize the effect of heparin. With its dimeric structure and its adhesion promoting properties, FGFRL1 resembles the nectins, a family of cell adhesion molecules found at cell-cell junctions.     

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5′–3′- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.     

Na/K-ATPase beta1-subunit associates with neuronal growth regulator 1 (NEGR1) to participate in intercellular interactions

Neuronal growth regulator 1 (NEGR1) is a GPI-anchored membrane protein that is involved in neural cell adhesion and communication. Multiple genome wide association studies have found that NEGR1 is a generic risk factor for multiple human diseases, including obesity, autism, and depression. Recently, we reported that Negr1^−/− mice showed a highly increased fat mass and affective behavior. In the present study, we identified Na/K-ATPase, beta1-subunit (ATP1B1) as an NEGR1 binding partner by yeast two-hybrid screening. NEGR1 and ATP1B1 were found to form a relatively stable complex in cells, at least partially co-localizing in membrane lipid rafts. We found that NEGR1 binds with ATP1B1 at its C-terminus, away from the binding site for the alpha subunit, and may contribute to intercellular interactions. Collectively, we report ATP1B1 as a novel NEGR1-interacting protein, which may help deciphering molecular networks underlying NEGR1-associated human diseases.     

Inhibition of proteolysis by the proform of eosinophil major basic protein (proMBP) requires covalent binding to its target proteinase

By proteolytic cleavage of insulin-like growth factor binding proteins, the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) is able to control the biological activity of insulin-like growth factors. PAPP-A circulates in pregnancy as a proteolytically inactive complex, disulfide bound to the proform of eosinophil major basic protein (proMBP). We here demonstrate that co-transfection of mammalian cells with PAPP-A and proMBP cDNA results in the formation of a covalent PAPP-A/proMBP complex in which PAPP-A is inhibited. Formation of the complex also occurs when PAPP-A and proMBP synthesized separately are incubated. Complex formation was monitored by Western blotting, and by using an immunoassay specific for the complex. Using mutagenesis, we further demonstrate that the complex forms in a specific manner and depends on the presence of two proMBP cysteine residues. Mutated proMBP, in which Cys-51 and -169 are replaced by serine, is unable to form the covalent complex with PAPP-A. Of particular interest, such mutated proMBP further lacks the ability to inhibit PAPP-A. For the first time, this conclusively demonstrates that proMBP is a proteinase inhibitor. We further conclude that proMBP inhibits PAPP-A in an unusual manner, not paralleled by other proteinase inhibitors of our knowledge, which requires proMBP to be covalently bound to PAPP-A by disulfide bonds. ProMBP binding to PAPP-A most likely either abrogates substrate access to the active site of PAPP-A or induces a conformational change in the structure of PAPP-A, as we, by further mutagenesis, were able to exclude that the inhibitory mechanism of proMBP is based on a cysteine switch-like mechanism.     

重组人生长激素治疗儿童特发性矮小症的疗效及对血清Ghrelin和IGF-1水平的影响

目的:探讨重组人生长激素(rhGH)治疗儿童特发性矮小症(ISS)的疗效及对血清饥饿激素(Ghrelin)、胰岛素样生长因子-1(IGF-1)水平的影响。方法:选取2014年1月-2016年8月期间我院收治的ISS患儿114例为研究对象。按照随机数字表法分为实验组(n=57)与对照组(n=57)。其中对照组给予常规治疗,实验组在对照组基础上联合rhGH治疗,两组疗程均为12个月。比较两组患儿的临床疗效,同时观察并对比两组患儿治疗前后血清Ghrelin以及IGF-1水平。结果:治疗后两组患儿身高、生长速率均较治疗前升高,且实验组高于对照组,差异有统计学意义(P0.05);治疗后两组患儿体重、总甲状腺素、骨龄、空腹血糖水平较治疗前比较差异无统计学意义(P0.05)。治疗后两组患儿血清Ghrelin水平较治疗前降低,且实验组低于对照组,血清IGF-1水平较治疗前升高,且实验组高于对照组,差异均有统计学意义(P0.05)。实验组不良反应发生率为5.26%,与对照组的0.00%比较,差异无统计学意义(P0.05)。结论:ISS患儿应用rhGH治疗效果满意,可明显改善ISS患儿体内血清IGF-1、Ghrelin水平,安全无副作用,促进患儿健康成长。     

Screening of human vascular endothelial growth factor (VEGF) receptor Flt-1 domain and study on its biological activity

Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1–2, 2–3 and 1–3 respectively were amplified from human placental cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system. The result showed that, not only loop 1–3, but also the smaller fragment loop 2–3 could bind to hVEGF₁₆₅. Recombinant expression plasmids pPIC9K/Flt-1(1–3) and pPIC9K/Flt-1(2–3) were constructed and transformed toPichia. pastoris host strain GS115, cultured in flasks, and expressed under the induction of 1% methanol. The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, its purity reached above 90%. Biological assayin vitro showed that the binding capacity of expressed soluble Flt-1 (2–3) to hVEGF₁₆₅ and its inhibiting effect on the proliferation of human umbilical veins endothelial cells (HUVEC) stimulated with hVEGF₁₆₅ were close to those of sFlt-1(1–3). Animal test showed that sFlt-1(2–3) could inhibit the formation of regenerate blood vessels stimulated with hVEGF₁₆₅ significantly.     

Screening of human vascular endothelial growth factor (VEGF) receptor Flt-1 domain and study on its biological activity

Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1-2, 2-3 and 1-3 respectively were amplified from human placen-tal cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system. The result showed that, not only loop 1-3, but also the smaller fragment loop 2-3 could bind to hVEGF165. Recombinant expression plasmids pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1 (2-3) were constructed and transformed to Pichia. pastoris host strain GS115, cultured in flasks, and expressed under the induction of 1 % methanol. The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, its purity reached above 90%. Biological assay in vitro showed that the binding capacity of expressed soluble Flt-1 (2-3) to hVEGF165 and its inhibiting effect on the proliferation of human um     

High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing the lowest PiT1 mRNA levels. The mechanism behind the role of PiT1 in increased cell proliferation is not known. We, however, found that compared to control cells, cultures of NIH3T3 cells overexpressing PiT1 upon seeding showed increased cell number after 24 h and had shifted more cells from G0/G1 to S+G2/M within 12 h, suggesting that an early event may play a role. We here show that expression of human PiT1 in NIH3T3 cells led to faster cell adhesion; this effect was not cell type specific in that it was also observed when expressing human PiT1 in MC3T3-E1 cells. We also show for NIH3T3 that PiT1 overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby contribute to their proliferative advantage within the first 24 h after seeding.     

Tyrosine kinase-independent activation of extracellular-regulated kinase (ERK) 1/2 by the insulin-like growth factor-1 receptor

The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gβγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase.     

Precursor of brain-derived neurotrophic factor (proBDNF) forms a complex with Huntingtin-associated protein-1 (HAP1) and sortilin that modulates proBDNF trafficking, degradation, and processing

proBDNF, a precursor of brain-derived neurotrophic factor (BDNF), is anterogradely transported and released from nerve terminals, but the mechanism underlying this process remains unclear. In this study, we report that proBDNF forms a complex with Huntingtin associated protein-1 (HAP1) and sortilin, which plays an important role in proBDNF intracellular trafficking and stabilization. The interaction of proBDNF with both HAP1A and sortilin in co-transfected HEK293 cells is confirmed by both fluorescence resonance energy transfer and co-immunoprecipitation. The frequent co-localization (>90%) of endogenous HAP1, sortilin, and proBDNF is also found in cultured cortical neurons. Mapping studies using GST pulldown and competition assays has defined the interacting region of HAP1 with proBDNF within amino acids 371-445 and the binding sequences of proBDNF to HAP1 between amino acids 65 and 90. Fluorescence recovery after photobleaching confirms the defective movement of proBDNF-containing vesicles in neurites of HAP1(-/-) neurons, which can be partially restored by reintroducing HAP1 cDNA into the neurons. However, the effect is significantly increased by simultaneously reintroducing both HAP1 and sortilin. proBDNF and HAP1 are highly co-localized with organelle markers for the Golgi network, microtubules, molecular motor, or endosomes in normal neurons, but this co-localization is reduced in HAP1(-/-) neurons. Co-immunoprecipitation and Western blot showed that sortilin stabilizes the proBDNF·HAP1 complex in co-transfected HEK293 cells, helping to prevent proBDNF degradation. Furthermore, the complex facilitates furin cleavage to release mature BDNF.     

表皮生长因子对人二倍体成纤维细胞p21~(WAF-1)基因表达的双向性影响

p2 1 WAF-1又称 sdi- 1 ,是细胞周期蛋白依赖性蛋白激酶 ( CDK)的抑制物基因 ,与细胞增殖调控及细胞衰老密切相关 .本研究为了解正常细胞中 p2 1 WAF-1对生长因子的反应性 ,以及其在细胞衰老时的表现 .我们以不同代龄的人胚肺二倍体成纤维细胞 ( 2 BS细胞株 )为实验对象 ,通过 Northern杂交术 ,观察表皮生长因子 ( EGF)对年轻 (低代龄 )细胞与衰老 (高代龄 )细胞 p2 1 WAF-1基因表达的影响 .结果显示 :p2 1 WAF-1在衰老 2 BS细胞中高表达 .EGF对年轻细胞 p2 1 WAF-1的表达有诱导作用 ,对衰老细胞有轻微诱导作用 ,在刺激后 3h左右达高峰 ,3～ 6h逐渐回落 ,并持续下降 .作用后 1 2h,其表达水平反而远低于作用前 .此作用在年轻细胞较为明显 .由此可见 :( 1 ) EGF对人二倍体成纤维细胞 p2 1 WAF-1的表达有双向性影响 ,先是一过性诱导 ,随后转为阻抑 ;( 2 )衰老细胞 p2 1 WAF-1对EGF的反应性有所降低     

Cadherin-related neuronal receptor 1 (CNR1) has cell adhesion activity with beta1 integrin mediated through the RGD site of CNR1

Cadherin-related neuronal receptor (CNR) proteins are a diverse set of synaptic protocadherins, but little is known about its adhesive properties. We found that overexpressed CNR1 protein localized on the cell surface of HEK293T cells and increased the calcium-dependent cell aggregation potential. However, we could not detect the strong homophilic binding activity of CNR1 EC-Fc fusion protein in vitro. Parental HEK293T cells adhered to Arg-Gly-Asp (RGD) motif of EC1 domain of CNR1-Fc fusion protein. The fusion protein that the Asp73 of EC1 point-mutated to Glu (RGE-Fc) lost the adhesive activity. The adhesion activity of HEK293T cells to CNR1 EC-Fc fusion protein was completely blocked by inhibitors of integrins, including RGDS peptide and anti-beta1 integrin antibodies. The increased cell-aggregative property of CNR1 transfectants was also blocked by RGDS peptides. At cell-cell junctions of the CNR1 transfectants, co-localization between CNR1 and HEK293T endogenous beta1 integrin was observed. Furthermore, the spatiotemporal expression patterns of CNR and beta1 integrin nearly overlapped in the molecular layer of the developing mouse cerebellum in the main stage of synaptogenesis. These results indicate that CNR1 has a heterophilic, calcium-dependent cell adhesion activity with the beta1 integrin subfamily, and raise the possibility of CNR-beta1 integrin association in synaptogenesis.     

血清PRDX1、FGF4、Hepc25水平与急性缺血性脑卒中患者病情及预后的关系研究

摘要 目的：探讨血清过氧化还原蛋白1（PRDX1）、成纤维细胞生长因子4（FGF4）、铁调素25（Hepc25）与急性缺血性脑卒中（AIS）患者病情严重程度和预后的关系。方法：选择2018年1月到2020年1月我院收治的73例AIS患者（AIS组）和同期于我院进行体检的56例健康者（对照组）,根据入院当日美国国立卫生研究院卒中量表（NIHSS）评分将AIS组进一步分为轻症组（NIHSS评分＜6分,21例）、中症组（6分≤NIHSS评分＜13分,38例）、重症组（NIHSS评分≥13分,14例）。根据患者出院后改良Rankin量表(mRS)评分将其分为预后不良组（≥3分,18例）和预后良好组（0～2分,55例）。检测所有受试者血清PRDX1、FGF4、Hepc25水平,分析PRDX1、FGF4、Hepc25与NIHSS、mRS评分相关性,分析AIS患者预后的影响因素。结果：AIS组血清PRDX1、FGF4、Hepc25水平均高于对照组（P＜0.05）,重症组血清PRDX1、FGF4、Hepc25水平高于中症组和轻度症组（P＜0.05）,且中症组血清PRDX1、FGF4、Hepc25水平高于轻症组（P＜0.05）,预后不良组血清PRDX1、FGF4、Hepc25水平高于预后良好组（P＜0.05）。AIS患者血清PRDX1、FGF4、Hepc25水平均与NHISS评分、mRS评分呈正相关（r=0.636、0.794、0.682;0.619、0.705、0.713,P＜0.05）。单因素分析结果显示年龄、高血压、糖尿病、入院时NIHSS评分与AIS患者预后有关（P＜0.05）,多因素Logistic回归分析结果显示入院时高NIHSS评分,高血清PRDX1、FGF4、Hepc25水平是AIS患者预后不良的危险因素（P＜0.05）。结论：AIS患者血清RDX1、FGF4、Hepc25水平明显升高,高水平RDX1、FGF4、Hepc25与AIS患者严重神经缺损和预后不良密切相关,可以作为AIS预后评估的辅助生物学指标。     

Synthesis and biological evaluation of a 3-positon epimer of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71)

1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ (ED-71), an analog of active vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], possesses a hydroxypropoxy substituent at the 2β-position of 1,25(OH)₂D₃. ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)₂D₃. Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)₂D₃, an epimer of 1,25(OH)₂D₃ at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)₂D₃ at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D₃ analogs were found to be 1,25(OH)₂D₃ > ED-71 ≥ 3-epi-1,25(OH)₂D₃  3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål)

To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella.     

Immunohistochemical study of vascular endothelial growth factor (VEGF), tumor suppressor protein (p53) and intercellular adhesion molecule (ICAM-1) in the conjunctiva of diabetic patients

Summary The expression pattern of VEGF, p53 and ICAM-1 was studied in conjunctiva of diabetic patients with and without retinopathy. All patients underwent a complete ophthalmic examination, including retinal fluorescein angiography. Indirect immunoperoxidase method was performed on 20 eyes of 20 patients with type II diabetes without DR and on 5 eyes of 5 patients with PDR. A control study was performed on 6 normal conjunctiva undertaken during cataract surgery. Immunoreactivity of VEGF, p53 and ICAM-1 was found in epithelial, fibroblast and vascular endothelial cells. For the same duration of diabetes, a strong to moderate or weak immunoreactivity was observed in the conjunctiva of patients without retinopathy. In patients with PDR, the expression was strong for all these proteins. The immunoreactivity was correlated between VEGF, p53 and ICAM-1. In the normal conjunctiva, a weak to negative immunostaining was observed. The presence of these proteins in the conjunctiva of diabetic patients without retinopathy may add new data in the pathogenesis of diabetic retinopathy. Further studies are needed to confirm this hypothesis.