Intact glycation end products containing carboxymethyl-lysine and glyoxal lysine dimer obtained from synthetic collagen model peptide

Advanced glycation end products (AGE) are accumulated in human tissues when long-lived proteins are glycated due to hyperglycemia and/or aging. In this study, we synthesized a collagen model peptide, Ac-(Pro-Hyp-Gly)(5)-Pro-Lys-Gly-(Pro-Hyp-Gly)(5)-Ala-NH(2) to investigate intact AGEs in peptides. The peptide formed a stable triple helix structure, and was subjected to glycation reactions with glucose, ribose and glyoxal. Besides carboxymethyl-lysine in the peptide, a conjugated form linked with glyoxal lysine dimer (GOLD) was detected upon treatment with glyoxal. This is the first example of intact glycation-derived dimers of peptides retaining intrinsic protein structures.     

Direct comparison of membrane interactions of model peptides composed of only Leu and Lys residues

We compared the properties of two peptides of identical size and amino acid composition, Ac-(LKKL)(5)-NHEt and Ac-(KL)(10)-NHEt. Both are amphipathic, but only Ac-(LKKL)(5)-NHEt is a potent promoter of negative curvature. CD studies performed in the presence of lipids confirmed that under these conditions Ac-(LKKL)(5)-NHEt forms an alpha-helix, and Ac-(KL)(10)-NHEt adopts a beta structure. We studied their binding affinity by centrifugation and isothermal titration calorimetry techniques. The Ac-(LKKL)(5)-NHEt bound to zwitterionic and anionic liposomes, while Ac-(KL)(10)-NHEt interacted mainly with anionic liposomes. Ac-(LKKL)(5)-NHEt was more lytic than Ac-(KL)(10)-NHEt for zwitterionic palmitoyloleoylphosphatidylcholine (POPC) liposomes, and for liposomes composed of lipids extracted from either sheep or human erythrocytes (RBC). Both peptides had similar lytic and lipid mixing activities for liposomes containing anionic lipids. Both peptides were highly hemolytic, with Ac-(LKKL)(5)-NHEt active against sheep RBC and Ac-(KL)(10)-NHEt more active against human RBC. From their respective minimal effective concentrations (MECs) as antimicrobial agents, we judged Ac-(KL)(10)-NHEt to be 2 to 5-fold more potent than Ac-(LKKL)(5)-NHEt in media that contained physiological concentrations of NaCl. Notwithstanding, both peptides had MECs <1 microg/mL for Escherichia coli and Pseudomonas aeruginosa and <4 microg/mL for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Although selectivity of antimicrobial peptides for bacterial membranes may result, in part, from the preferential display of anionic residues in these membranes, inability to interact with or bind to zwitterionic phospholipids offers no guarantee that the peptide will lack appreciable cytotoxicity for host cells.     

Glycosylation/Hydroxylation-induced stabilization of the collagen triple helix. 4-trans-hydroxyproline in the Xaa position can stabilize the triple helix

We have shown recently that glycosylation of threonine in the peptide Ac-(Gly-Pro-Thr)(10)-NH(2) with beta-d-galactose induces the formation of a collagen triple helix, whereas the nonglycosylated peptide does not. In this report, we present evidence that a collagen triple helix can also be formed in the Ac-(Gly-Pro-Thr)(10)-NH(2) peptide, if the proline (Pro) in the Xaa position is replaced with 4-trans-hydroxyproline (Hyp). Furthermore, replacement of Pro with Hyp in the sequence Ac-(Gly-Pro-Thr(beta-d-Gal))(10)-NH(2) increases the T(m) of the triple helix by 15.7 degrees C. It is generally believed that Hyp in the Xaa position destabilizes the triple helix because (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) form stable triple helices but the peptide (Hyp-Pro-Gly)(10) does not. Our data suggest that the destabilizing effect of Hyp relative to Pro in the Xaa position is only true in the case of (Hyp-Pro-Gly)(10). Increasing concentrations of galactose in the solvent stabilize the triple helix of Ac-(Gly-Hyp-Thr)(10)-NH(2) but to a much lesser extent than that achieved by covalently linked galactose. The data explain some of the forces governing the stability of the annelid/vestimentiferan cuticle collagens.     

Stabilization of triple-helical structures of collagen peptides containing a Hyp-Thr-Gly, Hyp-Val-Gly, or Hyp-Ser-Gly sequence

The single-crystal structures of three collagen-like host-guest peptides, (Pro-Pro-Gly)(4) -Hyp-Yaa-Gly-(Pro-Pro-Gly)(4) [Yaa = Thr, Val, Ser; Hyp = (4R)-4-hydroxyproline] were analyzed at atomic resolution. These peptides adopted a 7/2-helical structure similar to that of the (Pro-Pro-Gly)(9) peptide. The stability of these triple helices showed a similar tendency to that observed in Ac-(Gly-Hyp-Yaa)(10) -NH(2) (Yaa = Thr, Val, Ser) peptides. On the basis of their detailed structures, the differences in the triple-helical stabilities of the peptides containing a Hyp-Thr-Gly, Hyp-Val-Gly, or Hyp-Ser-Gly sequence were explained in terms of van der Waals interactions and dipole-dipole interaction between the Hyp residue in the X position and the Yaa residue in the Y position involved in the Hyp(X):Yaa(Y) stacking pair. This idea also explains the inability of Ac-(Gly-Hyp-alloThr)(10) -NH(2) and Ac-(Gly-Hyp-Ala)(10) -NH(2) peptides to form triple helices. In the Hyp(X):Thr(Y), Hyp(X):Val(Y), and Hyp(X):Ser(Y) stacking pairs, the proline ring of the Hyp residues adopts an up-puckering conformation, in agreement with the residual preference of Hyp, but in disagreement with the positional preference of X in the Gly-Xaa-Yaa sequence.     

The position of the LysN epsilon H2-grafted antigens along the sequential oligopeptide carrier, Ac-(Aib-Lys-Aib-Gly)n (SOCn-II), influences the antibody recognition: application to the Sm main autoimmune epitope

A sequential oligopeptide carrier of antigenic peptides is presented, incorporating two Aib residues in each repetitive moiety: Ac-(Aib-Lys-Aib-Gly)(n) (SOC(n) -II; n = 2-4). The conformational study, by (1)H-nmr, CD, and Fourier transform ir spectroscopy, indicated that the SOC(n) -II carrier displays a pronounced 3(10)-helix, compared to the Ac-(Lys-Aib-Gly)(n) (SOC(n) -I) carrier of the same approximately backbone length, previously reported. One of the dominant autoimmune epitopes of the Sm and U1RNP cellular components, the PPGMRPP sequence, was coupled to the Lys-N(epsilon)H(2) groups of the SOC(n) -II carrier and used as antigenic substrate for detecting anti-Sm/U1RNP autoantibodies in ELISA assays. Anti-Sm antibodies are highly specific for systemic lupus erythematosus, while anti-U1RNP are specific for mixed connective tissue disease. The anti-(PPGMRPP)(5)-SOC(n) -II ELISA was compared with the anti-(PPGMRPP)(n) -SOC(n) -I ELISA, provided that both antigenic substrates possess the same amount of the epitope replicates. The significance of the lysine positions along the oligopeptide backbone of the carrier for a favorable antibody recognition of the anchored antigens is also examined.     

Antiglycation effect of gliclazide on in vitro AGE formation from glucose and methylglyoxal

Gliclazide, a sulfonylurea widely used for treatment of diabetes mellitus, is known to scavenge reactive oxygen species. To clarify whether its antioxidative ability interferes with the glycation processes, we incubated bovine serum albumin (BSA) with 1 M glucose or 1 mM methylglyoxal, in the presence or absence of gliclazide, and observed the formation of advanced glycation end products (AGEs). AGE production was assessed by AGE-specific fluorescence, an enzyme-linked immunosorbent assay (ELISA), and Western blotting. The fluorescence at excitation/emission wavelengths of 320/383 nm and 335/385 nm was definitely increased by incubating BSA with 1 M glucose or 1 mM methylglyoxal, and 1 mM gliclazide significantly blunted the fluorescent augmentation, in both wavelengths, in a dose-dependent fashion. Gliclazide almost equaled to aminoguanidine, a putative antiglycation agent, in the inhibitory effect on the glucose-induced fluorescence, while the methylglyoxal-derived fluorescent formation was less suppressed by gliclazide than by aminoguanidine. The AGE concentrations determined by ELISA showed similar results. Incubation of BSA with 1 M glucose or 1 mM methylglyoxal yielded an apparent increase in carboxymethyllysine or argpyrimidine. Both AGEs were significantly lowered by 1 mM gliclazide and a reduction of glucose-derived carboxymethyllysine was comparable to that caused by aminoguanidine. The results of Western blotting supported the findings in ELISA. To our knowledge, the present study provides the first evidence of the antiglycation effect of gliclazide on in vitro AGE formation from glucose and methylglyoxal.     

Unique side chain conformation of a Leu residue in a triple-helical structure

Single crystal structures of host-guest peptides, (Pro-Hyp-Gly)(4)-Leu-Hyp-Gly-(Pro-Hyp-Gly)(5) (LOG1) and (Pro-Hyp-Gly)(4)- (Leu-Hyp-Gly)(2)-(Pro-Hyp-Gly)(4) (LOG2), have been determined at 1.6 A and 1.4 A resolution, respectively. In these crystals, the side chain conformations of the Leu residues were (+)gauche-trans. This conformational preference for the Leu side chain in the Leu-Hyp-Gly sequence was explained by stereochemical considerations together with statistical analysis of Protein Data Bank data. In the (+)gauche-trans conformation, the Leu side chain can protrude along the radial direction of the rod-like triple-helical molecule. One strong hydrophobic interaction of the Leu residue was observed between adjacent molecules in the LOG2 crystal. Because the Leu-Hyp-Gly sequence is one of the most frequently occurring triplets in Type I collagen, this strong hydrophobic interaction can be expected in a fibrillar structure of native collagen. All the Leu residues in the asymmetric unit of the LOG1 and LOG2 crystals had water molecules hydrogen bonded to their NH. These water molecules made three additional hydrogen bonds with the Hyp OH, the Gly O[double bond]C, and a water molecule in the second hydration shell, forming a tetrahedral coordination of hydrogen bonds, which allows a smaller mean-square displacement factor of this water oxygen atom than those of other water molecules. These hydrogen bonds stabilize the molecular and packing structures by forming one O[double bond]C(Gly)---W---OH(Hyp) intra-molecular linkage and two NH(Leu)---W---O[double bond]C(Gly) and NH(Leu)---W---OH(Hyp) inter-molecular linkages.     

Hydroxylation-induced stabilization of the collagen triple helix. Acetyl-(glycyl-4(R)-hydroxyprolyl-4(R)-hydroxyprolyl)(10)-NH(2) forms a highly stable triple helix

The collagen triple helix is one of the most abundant protein motifs in animals. The structural motif of collagen is the triple helix formed by the repeated sequence of -Gly-Xaa-Yaa-. Previous reports showed that H-(Pro-4(R)Hyp-Gly)(10)-OH (where '4(R)Hyp' is (2S,4R)-4-hydroxyproline) forms a trimeric structure, whereas H-(4(R)Hyp-Pro-Gly)(10)-OH does not form a triple helix. Compared with H-(Pro-Pro-Gly)(10)-OH, the melting temperature of H-(Pro-4(R)Hyp-Gly)(10)-OH is higher, suggesting that 4(R)Hyp in the Yaa position has a stabilizing effect. The inability of triple helix formation of H-(4(R)Hyp-Pro-Gly)(10)-OH has been explained by a stereoelectronic effect, but the details are unknown. In this study, we synthesized a peptide that contains 4(R)Hyp in both the Xaa and the Yaa positions, that is, Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) and compared it to Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2), and Ac-(Gly-4(R)Hyp-Pro)(10)-NH(2). Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) showed a polyproline II-like circular dichroic spectrum in water. The thermal transition temperatures measured by circular dichroism and differential scanning calorimetry were slightly higher than the values measured for Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2) under the same conditions. For Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2), the calorimetric and the van't Hoff transition enthalpy DeltaH were significantly smaller than that of Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). We postulate that the denatured states of the two peptides are significantly different, with Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) forming a more polyproline II-like structure instead of a random coil. Two-dimensional nuclear Overhauser effect spectroscopy suggests that the triple helical structure of Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) is more flexible than that of Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). This is confirmed by the kinetics of amide (1)H exchange with solvent deuterium of Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2), which is faster than that of Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). The higher transition temperature of Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2), can be explained by the higher trans/cis ratio of the Gly-4(R)Hyp peptide bonds than that of the Gly-Pro bonds, and this ratio compensates for the weaker interchain hydrogen bonds.     

Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease

Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N(epsilon)-(carboxymethyl)lysine (CML). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degrees C for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CML formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CML is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.     

Role of carbohydrate in stabilizing the triple-helix in a model for a deep-sea hydrothermal vent worm collagen

The glycopeptide Ac-(Gly-Pro-Thr(beta-Gal))(10)-NH(2) forms a collagen-like triple-helix. A (1)H NMR structural analysis is reported for the peptides Ac-(Gly-Pro-Thr)(n)-NH(2) and Ac-(Gly-Pro-Thr(beta-Gal))(n)-NH(2), where n = 1, 5, and 10. NMR assignments for the individual peptides are made using one- and two-dimensional TOCSY, ROESY, and NOESY experiments. The NMR and corroborating CD data show that Ac-(Gly-Pro-Thr)(n)-NH(2), n = 1, 5, or 10, as well as Ac-(Gly-Pro-Thr(beta-Gal))(n)-NH(2), n = 1 or 5 peptides are unable to form collagen-like triple-helical structures. Furthermore, the equilibrium ratio of cis to trans isomers of the Pro residues is unaffected by the presence of carbohydrate. For Ac-(Gly-Pro-Thr(beta-Gal))(10)-NH(2), the kinetics of amide (1)H exchange with solvent deuterium indicate a slow rate of exchange for both the Gly and the Thr amide. The data are thus consistent with a model in which the carbohydrate stabilizes the triple helix through an occlusion of water molecules and by hydrogen bonding but not through an influence on the cis to trans isomer ratio.     

Immunochemical detection of advanced glycosylation end products in vivo.

Reducing sugars react with protein amino groups to form a diverse group of protein-bound moieties with fluorescent and cross-linking properties. These compounds, called advanced glycosylation end products (AGEs), have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. Although several AGEs have been identified on the basis of de novo synthesis and tissue isolation procedures, the measurement of AGE compounds in vivo has remained difficult. As an approach to the study of AGE formation in vivo, we prepared polyclonal antiserum to an AGE epitope(s) which forms in vitro after incubation of glucose with ribonuclease (RNase). This antiserum proved suitable for the detection of AGEs which form in vivo. Both diabetic tissue and serum known to contain elevated levels of AGEs readily competed for antibody binding. Cross-reactivity studies revealed the presence of a common AGE epitope(s) which forms after the incubation of diverse proteins with glucose. Cross-reactive epitopes also formed with glucose 6-phosphate or fructose. These data suggest that tissue AGEs which form in vivo appear to contain a common immunological epitope which cross-reacts with AGEs prepared in vitro, supporting the concept that immunologically similar AGE structures form from the incubation of sugars with different proteins (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332). None of the known AGEs, such as 4-furanyl-2-furoyl-1H-imidazole, 1-alkyl-2-formyl-3,4-diglycosylpyrrole, pyrraline, carboxymethyllysine, or pentosidine, were found to compete for binding to anti-AGE antibody. These data further suggest that the dominant AGE epitope which forms from the reaction of glucose with proteins under native conditions is immunologically distinct from the structurally defined AGEs described to date.     

Inhibition of CYP2E1 leads to decreased advanced glycated end product formation in high glucose treated ADH and CYP2E1 over-expressing VL-17A cells

Background

In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure.

Methods

In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied.

Results

Inhibition of CYP2E1 with 10 μM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2 mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5 mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation.

Conclusions

Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1.

General significance

The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.     

Dendrimers based lysine and their "starburst" polymeric derivatives: prospects of use in compacting DNA and in vitro delivery of genetic constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-NH2 (D1) and (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-[Lys(Plm)]2-Ala-NH2 (D2), as well as the starburst polymeric derivatives of D1, (pVIm)8-D1 and (pLys)n-D1, containing poly(N-vinylimidazole) and polylysine chains bound at a single point to the dendrimer amino groups. The conditions of dendrimer-plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on C2C12 mouse myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

Asymmetry in the triple helix of collagen-like heterotrimers confirms that external bonds stabilize collagen structure

Heating and subsequent cooling mixtures of (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) peptides leads to formation of model heterotrimeric collagen helices that can be isolated by HPLC. These heterotrimeric collagen peptide helices are shown to be fundamentally unstable as denaturing then renaturing experiments result in heterotrimeric/homotrimeric mixtures.As the proportion of hydroxyproline-containing chains in the trimers increases, differential scanning calorimetry shows that the helix melting temperatures and denaturation enthalpies increasing non-linearly. Three types of Rich-Crick hydrogen bonds observed by NMR allow modelling of heterotrimeric structures based on published homotrimeric X-ray data. This revealed a small axial movement of (Pro-Hyp-Gly)(10) chains towards the C-terminal of the helix, demonstrating heterotrimeric asymmetry.     

Coordination abilities of the 1-16 and 1-28 fragments of beta-amyloid peptide towards copper(II) ions: a combined potentiometric and spectroscopic study

Stoichiometry, stability constants and solution structures of the copper(II) complexes of the (1-16H), (1-28H), (1-16M), (1-28M), (Ac-1-16H) and (Ac-1-16M) fragments of human (H) and mouse (M) beta-amyloid peptide were determined in aqueous solution in the pH range 2.5-10.5. The potentiometric and spectroscopic data (UV-Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the (Ac-1-16H) and (Ac-1-16M) peptides compared to the (1-16H) and (1-16M) fragments, respectively. The (Ac-1-16H) peptide forms the 3N [N(Im)(6), N(Im)(13), N(Im)(14)] complex in a wide pH range (5-8), while for the (Ac-1-16M) fragment the 2N [N(Im)(6), N(Im)(14)] complex in the pH range 5-7 is suggested. At higher pH values sequential amide nitrogens are deprotonated and coordinated to copper(II) ions. The N-terminal amino group of the (1-16) and (1-28) fragments of human and mouse beta-amyloid peptide takes part in the coordination of the metal ion, although, at pH above 9 the complexes with the 4N [N(Im), 3N(-)] coordination mode are formed. The phenolate -OH group of the Tyr(10) residue of the human fragments does not coordinate to the metal ion.     

Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides.

We made use of a planar lipid bilayer system to examine the action of synthetic basic peptides which model the prepiece moiety of mitochondrial protein precursors and have antibacterial activity against Gram-positive bacteria. The sequences of the peptides used were as follows: Ac-(Ala-Arg-Leu)3-NHCH3 (3(3], Ac-(Leu-Ala-Arg-Leu)2-NHCH3 (4(2], Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], Ac-(Leu-Leu-Ala-Arg-Leu)2-NHCH3 (5(2]. These peptides interacted differently with planar lipid bilayer membranes and membrane conductance increased by the formation of ion channels. The effects of the peptides on the macroscopic current-increase and on the probability of channel formation, at the single channel level were in the order of 4(3) greater than 4(2) approximately 5(2) much greater than 3(3), a finding which correlates with the antibacterial activity of these peptides. The micromolar (microM) order concentration at which the channel was formed resembles that causing antibacterial activity. Thus, the peptide antibacterial activity may occur through an increase in ion permeability of the bacterial membrane. The single-channel properties were investigated in detail using 4(3), the peptide with the highest ion channel-forming activity. Many types of channels were observed with respect to conductance (2-750 pS) and voltage dependency of gating. However, the channels were all cation-selective. These results suggest that the ion channels formed by peptide 4(3) may be able to take on a variety of conformations and/or assembly.     

Design and synthesis of cationic Aib-containing antimicrobial peptides: conformational and biological studies.

Development of antimicrobial peptides has attracted considerable attention in recent years due to the excessive use of antibiotics, which has led to multiresistant bacteria. Cationic amphiphilic Aib-containing peptide models Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 1-4, and sequential cationic polypeptides (Arg-X-Gly)(n), X = Ala, Val, Leu, were prepared and studied for their antimicrobial and hemolytic activity, as well as for their proteolytic stability. Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 2, 3 and the polypeptide (Arg-Leu-Gly)(n) exhibited significant antimicrobial activity, and they were nontoxic at their MIC values and resistant, in particular the Aib-peptide models, to enzymatic degradation. The conformational characteristics of the peptide models were studied by circular dichroism (CD). Structure-activity relationship studies revealed the importance of the amphipathic alpha-helical conformation of the reported peptides in inducing antimicrobial effects. It is concluded that peptide models comprising cationic amino acids (Arg), helicogenic and noncoding residues (Aib) and/or hydrophobic and helix-promoting components (Leu) may lead to the development of antimicrobial therapeutics.     

Detection of noncarboxymethyllysine and carboxymethyllysine advanced glycation end products (AGE) in serum of diabetic patients.

BACKGROUND: The advanced stage of the Maillard reaction, which leads to the formation of advanced glycation end products (AGE), plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. N(epsilon)-(carboxymethyl)lysine (CML) is thought to be an important epitope for many of currently available AGE antibodies. However, recent findings have indicated that a major source of CML may be by pathways other than glycation. A distinction between CML and non-CML AGE may increase our understanding of AGE formation in vivo. In the present study, we prepared antibodies directed against CML and non-CML AGE. MATERIALS AND METHODS: AGE-rabbit serum albumin prepared by 4, 8, and 12 weeks of incubation with glucose was used to immunize rabbits, and a high-titer AGE-specific antiserum was obtained without affinity for the carrier protein. To separate CML and non-CML AGE antibodies, the anti-AGE antiserum was subjected to affinity chromatography on a column coupled with AGE-BSA and CML-BSA. Two different antibodies were obtained, one reacting specifically with CML and the other reacting with non-CML AGE. Circulating levels of CML and non-CML AGE were measured in 66 type 2 diabetic patients without uremia by means of the competitive ELISA. Size distribution and clearance by hemodialysis detected by non-CML AGE and CML were assessed in serum from diabetic patients on hemodialysis. RESULTS: The serum non-CML AGE level in type 2 diabetic patients was significantly correlated with the mean fasting blood glucose level over the previous 2 months (r = 0.498, p < 0.0001) or the previous 1 month (r = 0.446, p = 0. 0002) and with HbA(1c) (r = 0.375, p = 0.0019), but the CML AGE level was not correlated with these clinical parameters. The CML and non-CML AGE were detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. The hemodialysis treatment did not affect the high-molecular-weight protein fractions. Although the low-molecular-weight peptide fractions (absorbance at 280 nm and fluorescence) were decreased by hemodialysis, there was no difference before and after dialysis in the non-CML AGE- and CML-peptide fractions (1.15 and 0.85 kD fractions). CONCLUSIONS: We propose that both CML and non-CML AGE are present in the blood and that non-CML AGE rather than CML AGE should be more closely evaluated when investigating the pathophysiology of AGE-related diseases.     

Sequence dependence of kinetics and morphology of collagen model peptide self-assembly into higher order structures

The process of self-assembly of the triple-helical peptide (Pro-Hyp-Gly)(10) into higher order structure resembles the nucleation-growth mechanism of collagen fibril formation in many features, but the irregular morphology of the self-assembled peptide contrasts with the ordered fibers and networks formed by collagen in vivo. The amino acid sequence in the central region of the (Pro-Hyp-Gly)(10) peptide was varied and found to affect the kinetics of self-assembly and nature of the higher order structure formed. Single amino acid changes in the central triplet produced irregular higher order structures similar to (Pro-Hyp-Gly)(10), but the rate of self-association was markedly delayed by a single change in one Pro to Ala or Leu. The introduction of a Hyp-rich hydrophobic sequence from type IV collagen resulted in a more regular suprastructure of extended fibers that sometimes showed supercoiling and branching features similar to those seen for type IV collagen in the basement membrane network. Several peptides, where central Pro-Hyp sequences were replaced by charged residues or a nine-residue hydrophobic region from type III collagen, lost the ability to self-associate under standard conditions. The inability to self-assemble likely results from loss of imino acids, and lack of an appropriate distribution of hydrophobic/electrostatic residues. The effect of replacement of a single Gly residue was also examined, as a model for collagen diseases such as osteogenesis imperfecta and Alport syndrome. Unexpectedly, the Gly to Ala replacement interfered with self-assembly of (Pro-Hyp-Gly)(10), while the peptide with a Gly to Ser substitution self-associated to form a fibrillar structure.     

Staggered molecular packing in crystals of a collagen-like peptide with a single charged pair

The crystal structure of the triple-helical peptide, (Pro-Hyp-Gly)(4)-Glu-Lys-Gly-(Pro-Hyp-Gly)(5) has been determined to 1.75 A resolution. This peptide was designed to examine the effect of a pair of adjacent, oppositely charged residues on collagen triple-helical conformation and intermolecular interactions. The molecular conformation (a 7(5) triple helix) and hydrogen bonding schemes are similar to those previously reported for collagen triple helices and provides a second instance of water mediated N--H . . . O==C interchain hydrogen bonds for the amide group of the residue following Gly. Although stereochemically capable of forming intramolecular or intermolecular ion pairs, the lysine and glutamic acid side-chains instead display direct interactions with carbonyl groups and hydroxyproline hydroxyl groups or interactions mediated by water molecules. Solution studies on the EKG peptide indicate stabilization at neutral pH values, where both Glu and Lys are ionized, but suggest that this occurs because of the effects of ionization on the individual residues, rather than ion pair formation. The EKG structure suggests a molecular mechanism for such stabilization through indirect hydrogen bonding. The molecular packing in the crystal includes an axial stagger between molecules, reminiscent of that observed in D-periodic collagen fibrils. The presence of a Glu-Lys-Gly triplet in the middle of the sequence appears to mediate this staggered molecular packing through its indirect water-mediated interactions with backbone C==O groups and side chains.