Synthesis and enzymic hydroxylation of protocollagen model peptide containing a hydroxyproline residue.
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ABSTRACT:
SUBMITTER: Kikuchi Y
PROVIDER: S-EPMC1177246 | biostudies-other | 1971 Oct
REPOSITORIES: biostudies-other
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Project description:1. Synthetic polymers of l-prolyl-l-prolylglycine of defined chain length, (Pro-Pro-Gly)(n), were found to be substrates for the enzyme protocollagen-proline hydroxylase, with optimum chain length n=5. Boiling the polymer (Pro-Pro-Gly)(15) increased its activity as a substrate but had no effect on (Pro-Pro-Gly)(5). 2. Protection of both or one of the N- and C-terminal groups made (Pro-Pro-Gly)(3) a better substrate, and collagenase digestion of hydroxylated tert.-pentyloxy-carbonyl-(Pro-Pro-Gly)(3) benzyl ester indicated that the central prolyl residues were the major points of hydroxylation. 3. The results suggest that the long-chain peptides are optimum substrates but that a triple-stranded structure is inhibitory for hydroxylation.
| S-EPMC1185139 | biostudies-other
Project description:The hydroxyproline-containing proteins (HCPs) among secretory and vacuolar proteins play important roles in growth and development of higher plants. Many hydroxyproline-rich glycoproteins (HRGPs), including Arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins (PRPs), are identified as HCPs by bioinformatics approaches. The experimental evidence for validation of novel proline hydroxylation sites is vital for understanding their functional roles. In this study, the 62 HCPs containing 114 hydroxyproline (O, Hyp) residues were identified, and it was found that hydroxylation of proline residues in the HCPs could either constitute attachment sites for glycans or have other biological function in rice. The glycomodules of AO, OA, OG, VO, LO, and OE were abundant in the 62 HCPs. Further analysis showed that the 22 of 62 HCPs contained both signal peptides and transmembrane domains, and the 19 HCPs only contained transmembrane domains, while 21 HCPs contained neither. This study indicated the feasibility of mass spectrometry-based proteomics combined with bioinformatics approaches for the large-scale characterization of Hyp sites from complex protein digest mixtures. Furthermore, the expression of AGPs in rice was detected by using ?-GlcY reagent and JIM13 antibody. The results displayed that the AGPs were widely distributed in different tissues and organs of rice, especially expressed highly in lateral root, pollen and embryo. In conclusion, our study revealed that the HCPs and Hyp residues in rice were ubiquitous and that these Hyps could be candidates for linking to glycans, which laid the foundation for further studying the functions of HCPs and hydroxylation of proline residues in rice.
| S-EPMC7427127 | biostudies-literature
Project description:The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.
| S-EPMC2583625 | biostudies-literature
Project description:(32)P-labelled phosphoglucomutase was digested with trypsin after denaturation and two peptides were isolated that contained the bulk of the radioactivity bound to peptides. Both peptides appeared to derive from an identical section of the molecule. Peptic and subtilisin digests of the tryptic peptides were prepared. The resulting radioactive peptides were purified and their sequences studied. The presence of a single serine [(32)P]phosphate residue was clearly established. Difficulties in purification and low yields, especially of the tryptic peptide, prevented exhaustive sequence studies, but a tentative sequence is proposed as:Ala-Ile-Gly-Gly-Ile-Ile-Leu-Thr-Ala-SerP-His-Asx-Pro-Gly-Gly-Pro-(Asx(2),Gly)-Phe-Gly-Ile-Lys(where SerP represents serine phosphate and Asx represents aspartic acid or asparagine). The results do not support the presence of two serine phosphate residues in the denatured enzyme, but confirm previous results of a unique sequence around a single serine phosphate residue.
| S-EPMC1186757 | biostudies-other
Project description:Identification of hydroxyproline-containing proteins and hydroxylation of proline in rice
2020-06-09 | PXD017798 | iProX
Project description:Background Endogenous oxalate synthesis contributes to calcium oxalate stone disease and is markedly increased in the inherited primary hyperoxaluria (PH) disorders. The incomplete knowledge regarding oxalate synthesis complicates discovery of new treatments. Hydroxyproline (Hyp) metabolism results in the formation of oxalate and glycolate. However, the relative contribution of Hyp metabolism to endogenous oxalate and glycolate synthesis is not known.Methods To define this contribution, we performed primed, continuous, intravenous infusions of the stable isotope [15N,13C5]-Hyp in nine healthy subjects and 19 individuals with PH and quantified the levels of urinary 13C2-oxalate and 13C2-glycolate formed using ion chromatography coupled to mass detection.Results The total urinary oxalate-to-creatinine ratio during the infusion was 73.1, 70.8, 47.0, and 10.6 mg oxalate/g creatinine in subjects with PH1, PH2, and PH3 and controls, respectively. Hyp metabolism accounted for 12.8, 32.9, and 14.8 mg oxalate/g creatinine in subjects with PH1, PH2, and PH3, respectively, compared with 1.6 mg oxalate/g creatinine in controls. The contribution of Hyp to urinary oxalate was 15% in controls and 18%, 47%, and 33% in subjects with PH1, PH2, and PH3, respectively. The contribution of Hyp to urinary glycolate was 57% in controls, 30% in subjects with PH1, and <13% in subjects with PH2 or PH3.Conclusions Hyp metabolism differs among PH types and is a major source of oxalate synthesis in individuals with PH2 and PH3. In patients with PH1, who have the highest urinary excretion of oxalate, the major sources of oxalate remain to be identified.
| S-EPMC6054332 | biostudies-literature
Project description:Arabinosylation of hydroxyproline (Hyp) is a post-translational modification often found in secreted peptide signals in plants. The physiological importance of this modification was highlighted by the finding that CLAVATA3 (CLV3), a key peptide signal for regulating the fate of stem cells in the shoot apical meristem in Arabidopsis, contains three l-arabinose residues linked via linear ?-1,2-linkages. However, understanding the functions and properties of arabinosylated peptides has been hindered by difficulties in synthesizing the complex arabinose chain. Here we report the stereoselective total synthesis of ?-1,2-linked triarabinosylated CLV3 peptide ([Ara3]CLV3). Chemically synthesized [Ara3]CLV3 restricted stem cell activity more effectively than did unmodified CLV3 peptide. Comparison of mono-, di- and triarabinosylated CLV3 glycopeptides revealed that the biological activity increased progressively as the arabinose chain length increased. Thus, the arabinose chain length of CLV3 is important for its biological activity. Nuclear magnetic resonance spectroscopy and nuclear Overhauser effect-based structure calculations further revealed the structural impact of the arabinose chain on peptide conformation. The arabinose chain of [Ara3]CLV3 extends toward the C-terminal end of the peptide, and its non-reducing end is positioned proximal to the peptide backbone. Consequently, the arabinose chain causes distinct distortion in the C-terminal half of the peptide in a highly directional manner. The established synthetic route of [Ara3]CLV3 will greatly contribute to our understanding of the biology and biochemistry of arabinosylated peptide signals in plants.
| S-EPMC3589827 | biostudies-other
Project description:Biodegradable hydrogels were synthesized by the click reaction of 4-arm azido-terminated PEG differing in molecular weight (2,100 and 8,800) and two alkyne-terminated peptides: [alkyne]-GFLGK-[alkyne] and ([alkyne]-GFLG)(2)K. The physical properties of in situ formed hydrogels were examined. The hydrogels were highly elastic as determined by rheological and microrheological studies. Swelling degree and enzymatic degradation by papain were dependent on the molecular weight of the PEG, but not the peptide. For PEG8800-based hydrogels, time-course analysis of degradation showed that the molecular weight of the soluble fraction quickly reached the PEG precursor value. These findings may guide future design of hydrogels with controllable mechanical properties and enzymatic degradability.
| S-EPMC4608387 | biostudies-literature
Project description:We prepared a series of peptide-like 14-membered macrocycles containing an imidazole-4,5-dicarboxylic acid scaffold by using known coupling reagents and protecting group strategies. Yields of the purified macrocycles were poor on average, yet seemingly independent of amino acid substitution or stereochemistry. The macrocycles retain some level of conformational variability as observed by both molecular modeling and X-ray crystallography. These macrocycles represent a new class of structures for further development and for future application in high-throughput screening against a variety of biological targets.
| S-EPMC6268944 | biostudies-literature
Project description:1. Oxidized (polymerized) histidine ammonia-lyase from Pseudomonas testosteroni was activated with dithiothreitol and the reduced disulphide-linked cysteine residues of the native enzyme were carboxymethylated with iodo[(14)C]acetate. 2. The activity of the carboxymethylated enzyme was similar to that of the polymerized form and approx. 15% of that of the fully reduced form. 3. A tryptic digest of the [(14)C]carboxymethylated enzyme contained only one radioactive peptide. 4. The amino acid sequence of this peptide was shown to be Gly-Leu-Leu-Asp-Gly-Ser-Ala-Ile-Asn-Pro-Ser-His-Pro-Asn-Cys- (CH(2)CO(2)H)-Gly-Arg. 5. These findings show that, during polymerization, the disulphide bonds are formed between identical regions of the enzyme, and that the cysteine residue involved is also the one required in the reduced state for full activity of the enzyme.
| S-EPMC1166157 | biostudies-other