Incorporation of conformationally constrained beta-amino acids into peptides.

The use of norbornene units to induce the formation of beta-sheet and beta-turn type structures in peptides is discussed. The norbornene unit is readily prepared by a desymmetrization reaction and is easily incorporated into a peptide chain. Depending upon the exact nature of the norbornene unit, it is possible to form structures which resemble parallel beta-sheets, antiparallel beta-sheets or beta-turns. Similar peptide analogues incorporating a cis-2-amino-cyclopropane carboxylic acid unit can also be prepared. As an illustration of the application of this chemistry, a short, asymmetric synthesis of conformationally constrained metalloprotease inhibitors is presented.     

In vitro and in silico studies of novel synthetic ACE-inhibitory peptides derived from Saccharomyces cerevisiae protein hydrolysate

The structure-function relation of YR-10 (YGKPVAVPAR) was investigated by synthesizing four structural analogs of that including YHR-10 (YGKHVAVHAR), GA-8 (GKPVAVPA), GHA-8 (GKHVAVHA), and PAR-3 (PAR). GA-8 (GKPVAVPA) was synthesized on the basis of simulated enzymatic gastrointestinal digestion performed by bioinformatics tools (expasy-peptide cutter). This study explains the molecular mechanisms for the interaction of synthetic peptides with ACE. The IC₅₀ values of each were 139.554 ± 2.3, 61.91 ± 1.2, 463.230 ± 3.56, 135.135 ± 2.1, 514.024 ± 5.86 µM, respectively. Results indicated that Pro replacement with His in YR-10 and GA-8 increased ACE inhibitory activity respectively, by 55.63% and 70.82%. Removal of Tyr and Arg from respectively N and C terminal positions of YR-10, following in silico simulated gastrointestinal digestion caused the 3.31 fold decrease in ACE inhibitory activity. YHR-10 showed the best docking poses, and GHA-8 exhibited interaction with Zn²⁺. Lineweaver–Burk plots of most active peptides suggest that they act as noncompetitive inhibitors against ACE.     

In vitro mucosal digestion of synthetic gliadin-derived peptides in celiac disease

Two celiac-active synthetic peptides derived from the A-gliadin structure corresponding to residues 8–19 (LQPQNPSQQQPQ) and to 11–19 were digestedin vitro with small intestinal mucosa from children with celiac disease in remission and from normal children. The products of digestion were separated into two fractions on the basis of M_r<400 and M_r>400 by gel permeation chromatography and subjected to amino acid analysis. After digestion of the dodecapeptide with celiac mucosa, 71±14% (molar) of the total digestion products remained in the M_r>400 fraction. Glutamine, proline, serine, and asparagine were the major amino acids present. Glutamine, proline, and leucine were the major amino acids in the M_r<400 fraction. The M_r>400 fraction from the celiac mucosal digestion of the nonapeptide was of similar composition to the corresponding fraction from the dodecapeptide and represented 78±15% of the total products. Digestion of the two peptides with normal mucosa gave lower amounts of products in the M_r>400 fraction, but they were of similar composition to the corresponding fractions from the celiac mucosal digestion. Peptides such as NPSQQQP and QNPSQQQ may be present in the M_r>400 fractions since glutamine and proline are present in the approximate ratio of 2∶1, respectively. The results indicate a defect in the mucosal digestion of peptides which are active in an animal model of celiac disease.     

Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site

Synthetic peptides including the gamma-carboxylation recognition site and acidic amino acids were compared as substrates for vitamin K-dependent gamma-carboxylation by bovine liver carboxylase. The 28-residue proPT28 (proprothrombin -18 to +10) and proFIX28 (pro-Factor IX -18 to +10) were carboxylated with a Km of 3 microM. The Vmax of proPT28 was 2-3 times greater than that of proFIX28. An analog of proFIX28 that contained the prothrombin propeptide had a Vmax 2-3-fold greater than an analog of proPT28 that contained the Factor IX propeptide. proFIX28/RS-1, based upon Factor IX Cambridge, proFIX28/RQ-4, based upon Factor IX Oxford 3, and proFIX28 had equivalent Km and Vmax values. Analogs of proPT28 containing Ala6-Glu7 or Glu6-Ala7 were carboxylated at equivalent rates. A peptide containing Asp6-Asp7 was carboxylated at a rate of about 1% of that of Glu carboxylation. Carboxylation of peptides containing Asp6-Glu7 and Glu6-Asp7 yielded results identical with peptides containing Ala6-Glu7 and Glu6-Ala7. Carboxymethylcysteine was not carboxylated when substituted for Glu6 in a peptide containing Asp7. These results indicate that the prothrombin propeptide is more efficient in the carboxylation process than is the Factor IX propeptide, but that both propeptides direct carboxylation; the gamma-carboxylation recognition site does not include residues -4 and -1; aspartic acid and carboxymethylcysteine are poor substrates for the carboxylase, but aspartic acid does not inhibit the carboxylation of adjacent glutamic acids.     

In vitro digestive stability of complexes between gliadin and synthetic blocking peptides

Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions.     

In vitro non-enzymatic glycosylation of myofibrillar proteins

• 1.1. Glycation is non-enzymatic modification of proteins by sugars in which not only structural but also biological properties of proteins are altered.
• 2.2. Our in vitro experiments show that incubation of myofibrillar proteins with ribose results in sugar attachment to proteins and at the same time myofibrillar ATPase activity is lowered.
• 3.3. DETAPAC, aminoguanidine and 2-mercaptoethanol all partially block myofibrillar protein glycation and ATPase activity is less inactivated.
• 4.4. The dependence of ATPase activity of myofibrils incubated with ribose on the amount of 2-mercaptoethanol present suggests that also modification of SH groups is involved in enzyme inactivation.
• 5.5. Electrophoretic studies revealed that heavy chains of myosin, actin, and tropomyosins are proteins which are mainly glycated in vitro.

     

Regulation of TRP channels by N-linked glycosylation

A subset of TRP channel proteins undergoes regulatory N-linked glycosylation. A glycosylation site in the first extracellular loop of TRPV5 is enzymatically cleaved by a secreted glucuronidase, indirectly regulating channel function. Members of the TRPC family share a similar site, although details about a regulatory role are lacking. A second conserved TRP channel glycosylation site is found immediately adjacent to the channel pore-forming loop; both TRPV1 and TRPV4--and perhaps other TRPV family members--are influenced by glycosylation at this site. N-linked glycosylation, and the dynamic regulation of this process, substantially impacts function and targeting of TRP channels.     

Mechanisms and principles of N-linked protein glycosylation

N-linked glycosylation, a protein modification system present in all domains of life, is characterized by a high structural diversity of N-linked glycans found among different species and by a large number of proteins that are glycosylated. Based on structural, functional, and phylogenetic approaches, this review discusses the highly conserved processes that are at the basis of this unique general protein modification system.     

Immunochemical studies of pancreatic colipase-lipase interaction employing immobilized synthetic peptides.

In view to study the possible participation of the sequence portions of colipase including or close to the free carboxyl groups at positions 15 and/or 72 to the binding with pancreatic lipase, we have used three synthetic peptides matching portions 8-16, 59-67 and 67-72 of the amino acid sequence. Polyclonal rabbit anticolipase immune serum, which cross-reacts with peptides in ELISA, was fractionated on columns of peptide coupled to Sepharose. Of the three fractions of antibodies, only that interacting with peptide 8-16 had the capacity to inhibit colipase-dependent lipase activity by specifically preventing the association of lipase with its protein cofactor previously bound to lipid. We conclude that the region spanning residues 8-16 of colipase is of importance for colipase-lipase interaction in the active complex formed at interface.     

In vitro binding of synthetic acylated lipid-associating peptides to high-density lipoproteins: effect of hydrophobicity

To measure the effect of hydrophobicity on the binding of model apoproteins to lipoproteins, we synthesized a 15 amino acid lipid-associating peptide (LAP) with acyl chains of various lengths (0-18 carbons) bound to the N-terminal amino acid through a peptide bond. The acylated LAPs preferentially bound to high-density lipoprotein (HDL) and were activators of lecithin:cholesterol acyltransferase. Circular dichroic spectra indicated that the LAP association with phospholipid was accompanied by increased alpha-helical structure. The LAPs self-associated in solution as judged from tryptophan fluorescence analysis. These characteristics, which are comparable to those of apolipoprotein A-I, were strongly dependent upon the acyl chain length of the LAPs. The equilibrium constants (Keq) for the association of LAPs to reassembled HDL were measured by equilibrium dialysis at several temperatures. At 37 degrees C, Keq increased by 3 orders of magnitude as the number of carbon units was increased from 0 to 16; there was a log-linear relationship between Keq and the acyl chain length. The free energy of association (delta Ga) decreased by a constant value for each methylene unit added to the acyl chain (0.35 kcal mol-1), clearly demonstrating a strict hydrophobic effect. This change of delta Ga was enthalpy rather than entropy driven. Our data show that, with all other parameters including putative alpha-helicity, sequence, and molecular weight being constant, the binding of a lipid-associating peptide to lipoprotein is governed by its hydrophobicity.     

Eliminating antibody polyreactivity through addition of N-linked glycosylation

Antibody polyreactivity can be an obstacle to translating a candidate antibody into a clinical product. Standard tests such as antibody binding to cardiolipin, HEp-2 cells, or nuclear antigens provide measures of polyreactivity, but its causes and the means to resolve are often unclear. Here we present a method for eliminating antibody polyreactivity through the computational design and genetic addition of N-linked glycosylation near known sites of polyreactivity. We used the HIV-1-neutralizing antibody, VRC07, as a test case, since efforts to increase VRC07 potency at three spatially distinct sites resulted in enhanced polyreactivity. The addition of N-linked glycans proximal to the polyreactivity-enhancing mutations at each of the spatially distinct sites resulted in reduced antibody polyreactivity as measured by (i) anti-cardiolipin ELISA, (ii) Luminex AtheNA Multi-Lyte ANA binding, and (iii) HEp-2 cell staining. The reduced polyreactivity trended with increased antibody concentration over time in mice, but not with improved overall protein stability as measured by differential scanning calorimetry. Moreover, glycan proximity to the site of polyreactivity appeared to be a critical factor. The results provide evidence that antibody polyreactivity can result from local, rather than global, features of an antibody and that addition of N-linked glycosylation can be an effective approach to reducing antibody polyreactivity.     

Effect of N-linked glycosylation on hepatic lipase activity

Hepatic lipase (HL) is a secretory protein synthesized in hepatocytes and bound to liver endothelium. Previous studies have suggested that HL N-linked glycans are required for catalytic activity. To directly test this hypothesis, Xenopus laevis oocytes were used to express native rat HL or HL lacking one or both N-linked glycosylation sites. The expressed and secreted native HL had an apparent molecular mass of 53 kDa, consistent with purified rat liver HL. The mutant lacking both glycosylation sites, while poorly secreted, had an apparent molecular mass of 48 kDa, the same size observed for HL after enzymatic removal of N-linked oligosaccharides. Mutants lacking one of the two sites were intermediate in size and showed reduced secretion. Each of these expressed and secreted proteins had full catalytic activity that was inhibited by antisera to rat HL. Thus, N-linked glycosylation of rat HL, while important to lipase secretion, is not essential for the expression of lipase activity.