Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 1. [21-Desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin

The C-terminal region of the A chain of insulin has been shown to play a significant role in the expression of the biological activity of the hormone. To further delineate the contribution of this segment, we have synthesized [21-desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin, in which the C-terminal amino acid residue of the A chain of insulin, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an amide and an isopropylamide, respectively. Both insulin analogues display biological activity, 14-15% for the unsubstituted amide analogue and 20-22% for the isopropylamide analogue, both relative to bovine insulin. In contrast, a [21-desasparagine-A]insulin analogue has been reported to display less than 4% of the activity of the natural hormone [Carpenter, F. (1966) Am. J. Med. 40, 750-758]. The implications of these findings are discussed, and we conclude that the A20-A21 amide bond plays a significant role in the expression of the biological activity of insulin.     

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 2. [21-Asparagine diethylamide-A]insulin

We have synthesized [21-asparagine diethylamide-A]insulin, which differs from the parent molecule in that the free carboxyl group of the C-terminal amino acid residue, asparagine, of the A chain moiety has been converted to a diethylamide group. The analogue displays equivalent potency in receptor binding and biological activity, 48% and 56%, respectively, relative to bovine insulin. In contrast, we have reported previously [Burke, G. T., Chanley, J. D., Okada, Y., Cosmatos, A., Ferderigos, N., & Katsoyannis, P. G. (1980) Biochemistry 19, 4547-4556] that [21-asparaginamide-A]insulin exhibits a divergence in these properties, ca. 60% in receptor binding and ca. 13% in biological activity. The disparity in the biological behavior of these analogues is discussed, and we ascribe the modulation of biological activity independent of receptor binding activity observed between these analogues to the difference in the negativity of the carbonyl oxygen of the A chain moiety C-terminal amino acid residue.     

Conformational correlation and coupled motion between residue A21 and B25 side chain observed in crystal structures of insulin mutants at position A21

The C-terminal residue of the insulin A chain is invariant and kept as asparagine in all known insulin molecules from hagfish through birds to mammals. To get information on the role of this conserved residue, which is still unclear, the three-dimensional structures of four human insulin mutants, A21 Asn-->Gly, A21 Asn-->Asp, A21 Asn-->Ala, and A21 Asn-->Gln DesB30, were determined by X-ray crystallography. The four mutants crystallize separately into two kinds (rhombohedral and cubic) of crystals. In the refined structures, conformational correlation and coupled motion between the A chain C-terminal residue A21 and the B25 side chain was observed, in contrast to the nearly unchanged general structures as compared with the native insulin structures in their respective crystals. A detailed analysis suggests that residue A21 can affect insulin receptor binding by interaction with the B25 side chain and the B chain C-terminal segment to assist the B25 side chain rearranging into the 'active' conformation.     

Structure and activity of insulin, XV[1-5]. Further evidence for the importance of arginine residue B22 in the activity of insulin. Semisyntheses of despentapeptide-(B23 - 30)-insulins varied in B22 using desnonapeptide-(B22 - 30)-insulin and tetrapeptides

Insulin hexamethyl ester was digested by trypsin. The resulting desoctapeptide-(B23 - 30)-insulin pentamethyl ester was purified. This compound was digested by carboxypeptidase B to remove the arginine residue B22 at the end of the B chain. Then the N-terminal amino groups of the remaining desnonapeptide-(B22 - 30)-insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the glutamic acid residue B21 of this product was coupled to the following synthetic tetrapeptide esters: Arg-Gly-Phe-Phe-OMe, Lys(Boc)-Gly-Phe-Phe-OMe, Orn(Boc)-Gly-Phe-Phe-OMe, Cit-Gly-Phe-Phe-OMe, Ala-Gly-Phe-Phe-OMe and Gly-Gly-Phe-Phe-OMe. The syntheses of these peptide esters are described. After removal of all protecting groups, despentapeptide-insulin (B22-Arg) and analogues of this product with variation in position B22 could be obtained. They were purified by column chromatography. The biological activities of these components were determined by the mouse fall test. In the case of despentapeptide insulin (C-terminus Arg-Gly-Phe-Phe), the activity rose to the expected value of 34%. The insulin variants with amino acid residues other than arginine in position B22 had much lower activities: with lysine 13%, with ornithine 12%, with citrulline 9%, with alanine 8% and with glycine 6%. Desnonapeptide-insulin by itself posses an activity of 3%. These results demonstrate once more the essential nature of arginine residue B22 for insulin activity.     

H-bonding maintains the active site of type 1 copper proteins: site-directed mutagenesis of Asn38 in poplar plastocyanin

Type I Cu proteins maintain a trigonal N2S coordination group (with weak axial ligation) in both oxidation states of the Cu2+/+ ion, thereby reducing the reorganization energy for electron transfer. Requirements for maintaining this coordination group were investigated in poplar plastocyanin (Pcy) by mutation of a conserved element of the type 1 architecture, an asparagine residue (Asn38) adjacent to one of the ligating histidines. The side chain of this asparagine forms an active site clasp via two H-bonds with the residue (Ser85) adjacent to the ligating cysteine (Cys84). In addition, the main chain NH of Asn38 donates an H-bond to the thiolate ligand. We have investigated the importance of these interactions by mutating Asn38 to Gln, Thr, and Leu. The mutant proteins are capable of folding and binding Cu2+, but the blue color fades; the rate of fading increases in the order Gln < Thr < Leu. The color is not restored by ferricyanide, showing that the protein is modified irreversibly, probably by oxidation of Cys84. The more stable mutants N38Q and N38T were characterized spectroscopically. The wild-type properties are slightly perturbed for N38Q, but N38T shows remarkable similarity to another type 1 Cu protein, azurin (Azu) from Pseudomonas aeruginosa. The Cu-S(Cys) bond is longer in Azu than in Pcy, and the NH H-bond to the ligating S atom is shorter. Molecular modeling suggests a similar effect for N38T because the threonine residue shifts toward Ser85 in order to avoid a steric clash and to optimize H-bonding. These results demonstrate that H-bonding adjacent to the type 1 site stabilizes an architecture which both modulates the electronic properties of the Cu, and suppresses side reactions of the cysteine ligand.     

Structure-activity relationships of globomycin analogues as antibiotics

Globomycin (1a), a signal peptidase II inhibitor, and its derivatives show potent antibacterial activity against Gram-negative bacteria. The synthesis and antimicrobial activity of novel globomycin analogues are reported. The hydroxyl group in the L-Ser residue was essential for the antimicrobial activity and the length of the alkyl side chain greatly influenced the activity. In addition, derivatives that had a modified cyclic core exhibited weak activity. One of the analogues showed a wider antimicrobial spectrum, effective against not only Gram-negative but also Gram-positive bacteria.     

21-arginine-A] insulin: a biologically active analog.

An analog of sheep insulin which differs from the parent molecule in that the C-terminal amino acid residue of the A chain, asparagine, is replaced by arginine, has been synthesized and isolated in highly purified form. The [Arg21] A chain of sheep insulin was synthesized by the fragment condensation approach and isolated as the S-sulfonated derivative. Conversion of the latter into the sulfhydryl form and interaction with the S-sulfonated B chain of bovine (sheep) insulin yielded [Arg21-A] sheep insulin, which was purified by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. The [Arg21-A] sheep insulin shows potencies of 10.5--12.5 IU/mg when assayed by the mouse convulsion method and 8.6 IU/mg by the radioimmunoassay method (cf. 23--25 IU/mg for the natural hormone). It has been suggested that in the insulin molecule the A21 asparagine participates in salt bridge- and hydrogen bond-forming interactions which are critical in the biological activity of the hormone. Although the [Arg21-A] analog still retains these interactions, it is only ca. 50% as active as the natural hormone. It is speculated that other factors than the above mentioned interactions come into play, which involve the side chain of the A21 amino acid residue and affect the biological activity of the hormone.     

Enhancing the activity of insulin at the receptor interface: crystal structure and photo-cross-linking of A8 analogues

The receptor-binding surface of insulin is broadly conserved, reflecting its evolutionary optimization. Neighboring positions nevertheless offer an opportunity to enhance activity, through either transmitted structural changes or introduction of novel contacts. Nonconserved residue A8 is of particular interest as Thr(A8) --> His substitution (a species variant in birds and fish) augments the potency of human insulin. Diverse A8 substitutions are well tolerated, suggesting that the hormone-receptor interface is not tightly packed at this site. To resolve whether enhanced activity is directly or indirectly mediated by the variant A8 side chain, we have determined the crystal structure of His(A8)-insulin and investigated the photo-cross-linking properties of an A8 analogue containing p-azidophenylalanine. The structure, characterized as a T(3)R(3)(f) zinc hexamer at 1.8 A resolution, is essentially identical to that of native insulin. The photoactivatable analogue exhibits efficient cross-linking to the insulin receptor. The site of cross-linking lies within a 14 kDa C-terminal domain of the alpha-subunit. This contact, to our knowledge the first to be demonstrated from the A chain, is inconsistent with a recent model of the hormone-receptor complex derived from electron microscopy. Optimizing the binding interaction of a nonconserved side chain on the surface of insulin may thus enhance its activity.     

Importance of aliphatic side-chain structure at positions 2 and 3 of the insulin A chain in insulin-receptor interactions.

In order to evaluate the cause of the greatly decreased receptor-binding potency of the naturally occurring mutant human insulin Insulin Wakayama ([LeuA3]insulin, 0.2% relative potency), we examined (by the semisynthesis of insulin analogues based on N alpha-PheB1,N epsilon-LysB29-bisacetyl-insulin) the importance of aliphatic side chain structure at positions A2 and A3 (Ile and Val, respectively) in directing the interaction of insulin with its receptor. Analogues bearing glycine, alanine, alpha-amino-n-butyric acid, norvaline, norleucine, valine, isoleucine, allo-isoleucine, threonine, tert-leucine, or leucine at positions A2 or A3 were assayed for their potencies in competing for the binding of 125I-labeled insulin to isolated canine hepatocytes, as were analogues bearing deletions from the A-chain amino terminus or the B-chain carboxyl terminus. Selected analogues were also analyzed by far-UV CD and absorption spectroscopy of Co2+ complexes. Our results identify that (a) Ile and Val serve well at position A2, whereas residues with other side chains (including those with straight chains, alternatively configured beta-branches, or a gamma-branch) exhibit relative receptor-binding potencies in the range 1-5%; (b) greater flexibility is allowed side-chain structure at position A3, with Ile, allo-Ile, alpha-amino-n-butyric acid, and tert-Leu exhibiting relative receptor-binding potencies in the range 11-36%; and (c) simultaneous replacements at positions A2 and A3, and deletions of the COOH-terminal domain of the insulin B chain in related analogues, yield cumulative effects. These findings are discussed with respect to a model for insulin-receptor interactions that involves a structure-orienting role for residue A2, the direct interaction of residue A3 with receptor, and multiple separately defined elements of structure and of conformational adjustment.     

Synthesis and evaluation of vancomycin and vancomycin aglycon analogues that bear modifications in the residue 3 asparagine

The synthesis and biological evaluation of a set of residue 3 analogues of vancomycin and its aglycon are described. These investigations follow from the promising biological activity of a protected and synthetically modified vancomycin aglycon analogue in which the asparagine side chain was modified to possess a nitrile, rather than a carboxamide. Although this modification typically was detrimental to antimicrobial activity, hydrophobic vancomycin aglycon analogues that lack a lipid anchor as well as the disaccharide are detailed that exhibit unusual potency against VanB, but not VanA, resistant bacteria.     

Side reaction of pyrenylalanine-peptides containing NG-tosylarginine during detosylation with hydrogen fluoride

Formation of by-products of pyrenylalanine-peptides was observed during the cleavage of a tosyl group in pyrenylalanine-peptides containing an Arg(Tos) residue with HF. NMR and fluorescence experiments showed that by-products were compounds in which a pyrenyl group was modified with a tosyl group(s). The side reaction was little suppressed by the addition of usual scavengers such as anisole, 1,2-ethanedithiol, thioanisole and p-cresol. Under the conditions used for the cleavage of N alpha-Boc group, i.e., trifluoroacetic acid or 4 M HCl in dioxane, the pyrenylalanine residue was stable. No side reaction was, furthermore, observed in the reductive cleavage with sodium in liquid ammonia. These results suggest that the side reaction may be due to an electrophilic attack of tosyl cations to the electron-rich pyrenyl group in the pyrenylalanine residue under the HF-mediated acidic conditions.     

A post-translational modification of nuclear proteins, N(G),N(G)-dimethyl-Arg, found in a natural HLA class I peptide ligand

Presentation of peptides derived from endogenous proteins by class I major histocompatibility complex molecules is essential both for immunological self-tolerance and induction of cytotoxic T-cell responses against intracellular parasites. Despite frequent and diverse post-translational modification of eukaryotic cell proteins, very few class I-bound peptides with post-translationally modified residues are known. Here we describe a natural dodecamer ligand of HLA-B39 (B*3910) derived from an RNA-binding nucleoprotein that carried N(G),N(G)-dimethyl-Arg. Although common among RNA-binding proteins, this modification was not previously known among natural class I ligands. The sequence of this peptide was determined by Edman degradation and electrospray ion trap mass spectrometry. The fragmentation pattern of the dimethyl-Arg side chain observed with this latter technique allowed us to unambiguously assign the isomeric form of the modified residue. The post-translationally modified ligand was a prominent component (1-2%) of the B*3910-bound peptide repertoire. The dimethyl-Arg residue was located in a central position of the peptide, amenable to interacting with T-cell receptors, and most other residues in the middle region of the peptide were Gly. These structural features strongly suggest that the post-translationally modified residue may have a major influence on the antigenic properties of this natural ligand.     

Synthesis of an insulin analogue embodying a strongly fluorescent moiety, [19-Tryptophan-A]insulin

As part of our aim to study the conformation of insulin in solution by time-resolved fluorescence spectroscopy, we have synthesized the analogue [19-Tryptophan-A]insulin. In this compound, the tyrosine residue at position 19 of the A-chain of insulin, one of the most strongly conserved residues in insulins from various species, is substituted with the strongly fluorescent tryptophan residue. [19-Tryptophan-A]insulin displays 4.1±1.9% of the potency of natural insulin in binding to the insulin receptor from rat liver plasma membranes, 5.0±2.3% in stimulating lipogenesis in rat adipocytes, and 75.7±4% of the potency of insulin in radioimmunoassay. In connection with our previous work, these data indicate that an aromatic side chain at position A¹⁹ of insulin seems necessary but not sufficient for high biological activity. We further conclude that in regard to the immunogenic determinants of insulin, tryptophan in position A¹⁹ is an essentially neutral substitution for tyrosine in that position, in sharp contrast to the situation with regard to biological activity.     

Synthesis and Anti‐HIV Activity of Guanine Modified Fluorinated Acyclic Nucleoside Phosphonate Derivatives

The preparation of an unprecedented series of nucleobase modified 3‐fluoro‐2‐(phosphonomethoxy)propyl (FPMP) acyclic nucleosides in both their (R) and (S) enantiomerically pure forms is described. The synthesis focuses on a Mitsunobu alkylation reaction to construct the C?N(9) bond between a chiral fluorinated side‐chain residue and 6‐ or 7‐modified guanine analogs. Prodrugs of FPMP‐7‐deazaguanine were also synthesized by derivatization of the corresponding phosphonic acid functionality with (bis)diamyl aspartate amidate groups, leading to moderate activity against human immunodeficiency virus type 1 (HIV‐1).     

News & Notes: Specificity of a Neutral Zn-Dependent Proteinase from Thermoactinomyces sacchari Toward the Oxidized Insulin B Chain

The proteolytic specificity of the neutral Zn-dependent proteinase from Thermoactinomyces sacchari was determined by analysis of the peptides obtained after incubation with the oxidized insulin B chain as a substrate. The enzyme is an endopeptidase with broad specificity. In total, 12 peptide bonds in the B chain of insulin were hydrolyzed. The major requirement is that a hydrophobic residue such as Leu, Val, or Phe should participate with the α-amino group in the bond to be cleaved. However, hydrolysis of bonds at the N-terminal side of His, Thr, and Gly was also observed. The peptide bond Leu 15–Tyr 16 in the oxidized insulin B chain, which is the major cleavage site for the alkaline microbial proteinases, is resistant to the attacks of the enzyme from Thermoactinomyces sacchari and other neutral proteinases. The proteolytic activity of the Zn-dependent proteinase from T. sacchari is different from those of other metalloendopeptidases from microorganisms. Received: 10 November 1999 / Accepted: 15 December 1999     

A D-pathway mutation decouples the Paracoccus denitrificans cytochrome c oxidase by altering the side-chain orientation of a distant conserved glutamate

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa(3) cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-A resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.     

Site-directed mutagenesis to fine-tune enzyme specificity

We have used a combination of a genetic selection and oligonucleotide-directed mutagenesis to introduce a series of amino acid replacements for a single residue into Escherichia coli glutaminyl-tRNA synthetase. The mutant enzymes mischarge supF tRNA(Tyr), with glutamine, to varying degrees depending on the polarity of the side chain introduced but apparently not depending on the size or shape of the side chain. These results indicate that repulsive charge-charge interactions may be important for specific recognition of nucleic acids by proteins and illustrate how a mutant, derived from genetic selection, may be further modified in activity by oligonucleotide-directed mutagenesis.     

Development of a process to manufacture PEGylated orally bioavailable insulin

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.     

文昌鱼胰岛素样肽的表达、纯化、鉴定及其多克隆抗体制备

为了深入研究胰岛素和胰岛素样生长因子1（IGF－1）的起源和进化以及结构与功能的关系。表达了胰岛素和IGF－1的祖先分子－－文昌鱼胰岛素样肽（ILP）。重组单链ILP的基因用化学方法合成（从cDNA推测的ILPB结构域的C端和A结构域的N端用Ala-Ala-Lys三肽连接起来,并钭B28Arg突变为Lys）,克隆到表达载体pVT102-U中,ILP在酿酒酵母中得到有效表达。发酵液经4步分离纯化,得到均一的单链ILP,经质谱测定分子量和氨基酸组成分析证明表达产物正确。通过Lys-C蛋白内切酶处理将重组单链ILP转化成双链形式。虽然双链ILP与人胰岛素受体没有结合活力。但圆二色性光谱显示它与胰岛素的结构非常相似,用表达的单链ILP免疫新西兰大白兔,获得了高滴度的多克隆抗体。