重组单链胰岛素的连接肽与其生物功能的关系

为研究重组单链胰岛素的生物活性与连接肽之间的关系,用基因定点突变的方法分别以二肽A-K,七肽A-A-A-A-A-A-K和十二肽A-A-A-A-A-A-A-A-A-A-A-K连接胰岛素的B30和A1,得到3个单链胰岛素分子PIP,[A]₅PIP和[A]₁₀PIP.它们的受体结合能力分别为胰岛素的0.14%, 14.3% 和11.1%, 体内生物活性与受体结合能力一致,而它们的促生长活性分别为胰岛素的17%,116.3%和38%.结果表明:(ⅰ)单链胰岛素也具有胰岛素的促代谢和促生长功能;(ⅱ)单链胰岛素与胰岛素受体的结合能力与连接肽的长度和氨基酸组成密切相关, 其受体结合能力随连接肽的改变,可由无到100%,进一步说明胰岛素与其受体结合时,B链C端远离A链N端是必需的;(ⅲ)单链胰岛素的促细胞生长能力也与连接肽长度和组成密切相关,且比其自身的促代谢能力强.     

一种重组抗胰蛋白酶单体人胰岛素突变体的设计、制备和鉴定

用缺口双链DNA的定向突变方法分别将胰岛素前体中B链第 2 2、2 8、2 9和 3 0位改变为Asp、Lys、Pro和Lys,酵母分泌表达的前体经胰蛋白酶直接酶切 ,得到重组 [B2 2Asp、B2 8Lys、B2 9Pro、B3 0Lys]人胰岛素。它与受体的结合能力约为猪胰岛素的 6% ,而体内生物活力保留 5 0 %。通过FPLC分子筛测定其自身结合能力 ,在生理条件下浓度达 10 -4mol/L时它以单体形式存在。作为可抗胰蛋白酶酶解的单体胰岛素类似物 ,它可能具有一定的应用前景     

一种人胰岛素类似物的制备、鉴定与活性研究

采用PCR方法,将人胰岛素分子B链B10位His突变为Glu,在B25和B26位之间插入Glu,构建了［B10 Glu,B25-Glu-B26］胰岛素原融合蛋白基因.利用通用型质粒pBV220构建表达载体,在大肠杆菌DH5α中表达,表达蛋白为包含体形式,约占菌体总蛋白的20%～30%.经过复性、凝胶过滤等步骤得到胰岛素原融合蛋白.用胰蛋白酶和羧肽酶B酶切,经过DEAE离子交换和RP-HPLC纯化得到胰岛素突变体类似物,并经过质谱测定鉴定.凝胶过滤法测定了蛋白质分子自身的缔合性质,圆二色谱（CD）测定了构象的变化.并分别测定了放免活性、受体结合活性及小白鼠低血糖惊厥实验.结果表明,突变体分子缔合性明显下降.放免活性和受体结合活性分别约为标准胰岛素的63.5%和114.4%, 整体活力略高于天然胰岛素.     

明日叶查尔酮对2型糖尿病大鼠红细胞胰岛素受体亲和力的影响

目的:研究明日叶查尔酮对2型糖尿病大鼠红细胞胰岛素受体亲和力的影响.方法:将高脂喂养加链脲佐菌素注射诱发的2型糖尿病大鼠随机分为4组,每组10只.糖尿病对照组和高、中、低剂量组喂饲高脂饲料分别经口灌胃明日叶查尔酮0、30、10、5mg (kg·bw)-1,正常对照组为正常大鼠喂饲普通饲料,连续4周.测定空腹血糖、血清胰岛素与MDA、红细胞胰岛素受体结合常数与结合容量等指标.结果:高剂量组的高亲和力与低亲和力胰岛素受体结合常数高于糖尿病对照组,血糖、胰岛素和MDA含量则降低,差异均有显著性(P＜0.05).结论:明日叶查尔酮能提高2型糖尿病大鼠红细胞胰岛素受体亲和力,改善胰岛素抵抗.     

A8—10替换的胰岛素似物的合成及生物活性

本文报道了用Ｆｍｏｅ固相法合成３种胰岛素Ａ链小环（Ａ８－１０）被不同碱性氨基酸取代的Ａ链类似物,并分别与天然胰岛素Ｂ链重组成相应胰岛素类似物;经受体结合,整体活性及抗体结合实验,均表现出相应的活性。从中可以推测出：Ａ链小环区域不是胰岛素表现生物活性的重要部位,而是胰岛素与其抗体结合较重要的区域。     

A_（8—10）替换的胰岛素类似物的合成及生物活性

本文报道了用Ｆｍｏｃ固相法合成３种胰岛素Ａ链小环（Ａ８－１０）被不同碱性氨基酸取代的Ａ链类似物,并分别与天然胰岛素Ｂ链重组成相应胰岛素类似物;经受体结合,整体活性及抗体结合实验,均表现出相应的活性。从中可以推测出：Ａ链小环区域不是胰岛素表现生物活性的重要部位,而是胰岛素与其抗体结合较重要的区域。     

去B链羧端七肽胰岛素的免疫活性及受体结合能力

测定了胰岛素类似物去B 链羧端七肽胰岛素(DHPI)的免疫活性和它与受体的结合能力。DHPI 能与豚鼠抗胰岛素血清反应,生成免疫沉淀条纹。经放射免疫测定,它的免疫活力是胰岛素的1.8%。DHPI 在低剂量时不能和胰岛素受体(大白鼠脂肪细胞和豚鼠红细胞)结合,高剂量时有明显结合。DHPI 与豚鼠红细胞胰岛素受体的结合不显示负协同效应,而胰岛素与它的结合显示负协同效应。     

去B链羧端七肽胰岛素的免疫活性及受体结合能力

测定了胰岛素类似物去B链羧端七肽胰岛素(DHPI)的免疫活性和它与受体的结合能力。DHPI能与豚鼠抗胰岛素血清反应,生成免疫沉淀条纹。经放射免疫测定,它的免疫活力是胰岛素的1.8%。DHPI在低剂量时不能和胰岛素受体(大白鼠脂肪细胞和豚鼠红细胞)结合,高剂量时有明显结合。DHPI与豚鼠红细胞胰岛素受体的结合不显示负协同效应,而胰岛素与它的结合显示负协同效应。     

胰岛素作用原理的研究——Ⅰ.胰岛素及其类似物与其受体的相互作用

研究了一些胰岛素B链羧端肽段改变了的类似物与肝细胞及脂肪细胞的受体蛋白结合性质。这些结果说明,去B链羧端五肽胰岛素与胰岛素有相同的结合能力;去B链羧端六肽胰岛素及B_(23)被D-丙氨酸取代的去B链羧端六肽胰岛素有明显的结合;去B链羧端八肽胰岛素具极微的结合能力;胰岛素A链、促肾上腺皮质素、增血糖素不与胰岛素受体蛋白结合。在此基础上还讨论了B_(24)芳香环参与胰岛素结合部位的可能性,并假设B_(12),B_(16),B_(24),B_(25)疏水平面可能是结合部位的重要内容。而B_(20)～B_(23)U形转折及A_(21)…B_(22)盐键的作用是稳定疏水平面结构。胰岛素与受体的结合类似于胰岛素二体中两个单体的结合。     

胰岛素类似物的内源萤光光谱

为了进一步了解胰岛素构象与功能的关系,测定了不同条件下胰岛素和它的类似物的内源萤光光谱,胰岛素类似物包括去B链羧端五肽胰岛素,去B链羧端七肽胰岛素和去B链羧端八肽胰岛素。它们的发射光谱的峰在306nm,激发光谱峰在277nm。不同pH值,不同浓度的十二烷基硫酸钠和温度变化对胰岛素的荧光光谱的影响和对类似物的影响非常相似。说明这些类似物的环境酪氨酸残基的微环境是相似的。由于去五肽胰岛素有接近于天然胰岛素的生物活力,去七肽胰岛素没有生物活力,因此说明,胰岛素分子的受体结合部位中,B_(24～25)两个苯丙氨酸侧链本身具有重要的地位。并不是因为失去它们后引起胰岛素构象变化而失活的。     

Shortened insulin analogues: marked changes in biological activity resulting from replacement of TyrB26 and N-methylation of peptide bonds in the C-terminus of the B-chain

The role of three highly conserved insulin residues PheB24, PheB25, and TyrB26 was studied to better understand the subtleties of the structure-function relationship between insulin and its receptor. Ten shortened insulin analogues with modifications in the beta-strand of the B-chain were synthesized by trypsin-catalyzed coupling of des-octapeptide (B23-B30)-insulin with synthetic peptides. Insulin analogues with a single amino acid substitution in the position B26 and/or single N-methylation of the peptide bond at various positions were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by two types of in vitro assays, i.e., by the binding to the receptor of rat adipose plasma membranes and by the stimulation of the glucose transport into the isolated rat adipocytes. From our results, we can deduce several conclusions: (i) the replacement of tyrosine in the position B26 by phenylalanine has no significant effect on the binding affinity and the stimulation of the glucose transport of shortened analogues, whereas the replacement of TyrB26 by histidine affects the potency highly positively; [HisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide and [NMeHisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide show binding affinity 529 and 5250%, respectively, of that of human insulin; (ii) N-methylation of the B24-B25 peptide bond exhibits a disruptive effect on the potency of analogues in both in vitro studies regardless the presence of amino acid in the position B26; (iii) N-methylation of the B23-B24 peptide bond markedly reduces the binding affinity and the glucose transport of respective analogue [NMePheB24]-des-tetrapeptide (B27-B30)-insulin-B26-amide.     

Semisynthetic des-(B27-B30)-insulins with modified B26-tyrosine

Semisynthetic des-(B27-B30)-insulins containing modified B26-tyrosine residues were prepared to refine the understanding of the importance of position B26 with regard to biological and structural properties of the hormone. The following shortened insulin analogues were synthesized by trypsin-catalysed peptide-bond formation between the C-terminal amino acid ArgB22 of des-(B23-B30)-insulin and synthetic tetrapeptides as amino components: des-(B27-B30)-insulin, des-(B27-B30)-insulin-B26-methyl ester, -B26-carboxamide with varying C-terminal hydrophobicity of the B-chain, and [Tyr(NH2)B26]-, [Tyr(NO2)B26]-, [Tyr(I2)B26]-, [D-TyrB26]des-(B27-B30)-insulin-B26-carboxamide containing non-proteinogenic amino acids in position B26. Starting from insulin and an excess of synthetic Gly-Phe-Phe-Tyr-OMe as nucleophile, des-(B27-B30)-insulin-B26-methyl ester--the formal transpeptidation product at ArgB22--was formed in one step. Biological in vitro properties (binding to cultured human IM-9 lymphocytes, relative lipogenic potency in isolated rat adipocytes) of all semisynthetic analogues are reported, ranging from slightly decreased to two-fold receptor affinity and nearly three-fold biopotency relative to insulin. If the C-terminal tetrapeptide B27-B30 is removed, full relative insulin activity is still preserved, while the shortening results in the loss of ability to associate in solution. Only after carboxamidation or methyl esterification of TyrB26 the self-association typical of native insulin can be observed, and the CD-spectral effects in the near UV spectrum related to association and hexamerization of the native hormone are qualitatively reestablished. The results of this investigation underline the importance of position B26 to the modulation of hormonal properties and solution structure of the shortened insulins.     

The solution structure of a superpotent B-chain-shortened single-replacement insulin analogue

This paper reports on an insulin analogue with 12.5-fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy. The analogue is [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. C-terminal truncation of the B-chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of D-Ala for the wild-type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild-type hormone when the B-chain is present in full length. The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the "active" conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three-dimensional structure of [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface.     

Insulin analogues with modifications at position B26. Divergence of binding affinity and biological activity

In this study, we prepared several shortened and full-length insulin analogues with substitutions at position B26. We compared the binding affinities of the analogues for rat adipose membranes with their ability to lower the plasma glucose level in nondiabetic Wistar rats in vivo after subcutaneous administration, and also with their ability to stimulate lipogenesis in vitro. We found that [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2 were very potent insulin analogues with respect to their binding affinities (214 and 465%, respectively, compared to that of human insulin), but they were significantly less potent than human insulin in vivo. Their full-length counterparts, [NMeHisB26]-insulin and [NMeAlaB26]-insulin, were less effective than human insulin with respect to binding affinity (10 and 21%, respectively) and in vivo activity, while [HisB26]-insulin exhibited properties similar to those of human insulin in all of the tests we carried out. The ability of selected analogues to stimulate lipogenesis in adipocytes was correlated with their biological potency in vivo. Taken together, our data suggest that the B26 residue and residues B26-B30 have ambiguous roles in binding affinity and in vivo activity. We hypothesize that our shortened analogues, [NMeHisB26]-DTI-NH 2 and [NMeAlaB26]-DTI-NH 2, have different modes of interaction with the insulin receptor compared with natural insulin and that these different modes of interaction result in a less effective metabolic response of the insulin receptor, despite the high binding potency of these analogues.     

Shortened insulin with enhanced in vitro potency

After it has been shown that removal of residues B26-B30 leaves insulin with full biological activity, provided the new C-terminus is amidated (Fischer et al. (1985) Biol. Chem. Hoppe-Seyler 366, 521-525), it is demonstrated here that it does not even preclude enhancement of potency. 7 analogues of des-(B26-B30)-insulin-B25-amide were prepared by trypsin-mediated semisynthesis, the replacements being D-PheB24; HisB25, D-PheB25, TrpB25, TyrB25; D-PheB24,B25 and D-PheB24, TyrB25. Mere conversion of the configuration of B25-phenylalanine reduces in vitro potency to 0.5%. If B25-phenylalanine is, however, substituted by histidine or tyrosine activity is increased to 310 or 230, respectively. According to the features common to these two side chains, the favourable effect should be due to their ring structure with balanced aromatic and polar or H-bonding properties, respectively. The results indicate that in the complete insulin molecule the C-terminal pentapeptide modulates the subtle role that residues B24 and/or B25 play in receptor binding and activity; its presence may have a positive or negative effect. The drastic differences in activity between the shortened analogues are in no ways reflected in the CD spectra which are very similar, though clearly different from that of native insulin.     

Four new monomeric insulins obtained by alanine scanning the dimer-forming surface of the insulin molecule

The residues A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr and B27Thr, buried in the dimer of insulin, were identified by means of alanine-scanning mutagenesis. The receptor binding activity, in vivo biological potency and self-association properties of the seven single alanine human insulin mutants were determined. Four of the seven single alanine mutants, [B12Ala]human insulin, [B16Ala]human insulin, [B24Ala]human insulin and [B26Ala]human insulin, are monomeric insulin, which indicates that B12Val, B16Tyr, B24Phe and B26Tyr are crucial for the formation of insulin dimer. The monomeric [B16Ala]human insulin and [B26Ala]human insulin retain 27 and 54% receptor binding activity, respectively, and nearly the same in vivo biological potency compared with native insulin, so they could be developed as the fast-acting insulin.     

Chemical synthesis of [des(tetrapeptide B27--30), Tyr(NH2)26-B] and [des(pentapeptide B26--30), Phe(NH2)25-B] bovine insulins.

Two analogs of bovine insulin, [des(tetrapeptide B27--30), Tyr(NH2)26-B] and [des(pentapeptide B26--30), Phe(NH2)25-B] insulin, which differ from the parent molecule in that the C-terminal tetrapeptide and pentapeptide sequences, respectively, from the B chain have been eliminated and the newly exposed residues are amidated, have been synthesized. The [des(tetrapeptide B27--30), Tyr(NH2)26-B] insulin shows potencies of 16.8 IU/mg by the mouse convulsion assay method and 10.8 IU/mg by the radioimmunoassay method. The [des(pentapeptide B26--30), Phe(NH2)25-B] insulin possesses a potency of 10.5 IU/mg when assayed by the mouse convulsion method and 14 IU/mg by the radioimmunoassay technique. The potencies of these analogs are higher than the potencies of the respective non-amidated derivatives (Katsoyannis et al., 1973, 1974). It is speculated that the gradual decline of biological activity observed as amino acid residues are eliminated from the C-terminal region of the B chain of insulin is due to the proximity of a hydrophilic carboxyl group to the hydrophobic core of the protein molecule.     

The use of Fmoc-Lys(Pac)-OH and penicillin G acylase in the preparation of novel semisynthetic insulin analogs.

In this paper, we present the detailed synthetic protocol and characterization of Fmoc-Lys(Pac)-OH, its use for the preparation of octapeptides H-Gly-Phe-Tyr-N-MePhe-Thr-Lys(Pac)-Pro-Thr-OH and H-Gly-Phe-Phe-His-Thr-Pro-Lys(Pac)-Thr-OH by solid-phase synthesis, trypsin-catalyzed condensation of these octapeptides with desoctapeptide(B23-B30)-insulin, and penicillin G acylase catalyzed cleavage of phenylacetyl (Pac) group from Nepsilon-amino group of lysine to give novel insulin analogs [TyrB25, N-MePheB26,LysB28,ProB29]-insulin and [HisB26]-insulin. These new analogs display 4 and 78% binding affinity respectively to insulin receptor in rat adipose membranes.     

Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation

We have investigated (by use of semisynthetic insulin analogs and isolated canine hepatocytes) the role of invariant residue PheB24 in determining the affinity of insulin-receptor interactions. Our results confirm that replacement of PheB24 by D-Phe is not detrimental to ligand binding to receptor, show that D-Ala is well tolerated at position B24 (whereas Ala is not), and demonstrate that [GlyB24]insulin retains as much as 78% of the receptor binding potency of native insulin. Additional findings show that replacement of PheB24 by D-Pro or by alpha-aminoisobutyric acid results in analogs with severely decreased binding potency, and that the COOH-terminal domain containing residues B26-B30 plays a positive role in determining receptor binding potency in GlyB24-substituted insulin (whereas it plays a negative role in determining the receptor binding potency of its GlyB25-substituted counterpart). We interpret our results as identifying (a) a critical role for the insulin main chain near residue B24 in determining the affinity of receptor for ligand, (b) the importance of main chain flexibility in achieving a high affinity state of receptor-bound hormone, and (c) a potential interaction of the PheB24 side chain with receptor which initiates main chain structural changes in the natural hormone, but which does not itself confer affinity to ligand-receptor interactions.     

A shortened insulin with full in vitro potency

Des[(B26-30)-pentapeptide]insulin-B25-amide was prepared from protected des-[(B23-30)-octapeptide]insulin (pig) and H-Gly-Phe-Phe-NH2 by trypsin-mediated semisynthesis in a yield of 9% (based on insulin). The analogue was characterized with respect to chemistry, biological function and CD spectroscopy. While des[(B26-30)-pentapeptide]insulin with free carboxylate group exhibited a typical insulin activity of only 25% in vitro, des[(B26-30)-pentapeptide]insulinamide was fully active. Therefore des[(B26-30)-pentapeptide]insulin meets all structural and dynamic requirements for recognition and binding of the receptor as well as exertion of the biological effect, provided that the negative charge in the hydrophobic environment of PheB25 is neutralized.