RP–HPLC–ESI–MS characterization of novel peptide fragments related to rat parotid secretory protein in parasympathetic induced saliva

Two peptides (MW 1211.7 and 928.5 Da) were detected by RP–HPLC–ESI–MS analysis of parotid saliva secreted upon continuous parasympathetic stimulation. The peptide with the higher mass (PSPFr‐A) corresponded to the N‐terminal dodecapeptide (Fragment 1–12) of rat parotid secretory protein (PSP), while the peptide with the lower mass (PSPFr‐B) corresponded to the 4–12 fragment of the same protein. During stimulation, the PSPFr‐A secretion increased, while the PSPFr‐B secretion decreased (HPLC–ESI–MS). In the presence of cycloheximide, PSPFr‐A was not demonstrated, while the PSPFr‐B secretion decreased. In the presence of aprotinin, the PSPFr‐B secretion was almost abolished, while the PSPFr‐A secretion increased to higher levels than those observed in the absence of the inhibitor. In vitro perfusion, with artificial solution, of stimulated rat parotid glands excluded that the fragments were derived from the circulation. Neither peptide occurred in enriched granule preparations from unstimulated glands. The results suggest that at least two pathways – granular and vesicular – are responsible for the generation of the two peptides. PSPFr‐A is the first cleavage product in both pathways. PRPFr‐B is probably generated from granular PSPFr‐A only and, at the end of the granule mediated pathway, by the action of an enzyme of the serine protease class.     

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins.

The proteins in human parotid saliva have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into 20 or more well resolved species. The Coomassie Brilliant Blue (CBB) R-250 and silver staining procedures have been modified to overcome the problems encountered with staining of proline-rich proteins. By means of the CBB R-250 procedure which stains proline-rich proteins pink-violet, immunoblotting, concanavalin A binding, periodate-Schiff staining and zinc binding, all of the major proteins have been characterised. Substantial individual-to-individual differences were observed in the protein patterns formed. Comparison of parotid, submandibular, and whole saliva from a single individual indicated that fewer proline-rich proteins are expressed in submandibular saliva than in parotid, but whole saliva contains much lower levels than either duct secretion. The results will form a useful base for future research into the functions of salivary proteins.     

Sequence-specific recognition and cooperative dimerization of N-terminal aromatic peptides in aqueous solution by a synthetic host

This article describes the selective recognition and noncovalent dimerization of N-terminal aromatic peptides in aqueous solution by the synthetic host compound, cucurbit[8]uril (Q8). Q8 is known to bind two aromatic guests simultaneously and, in the presence of methyl viologen, to recognize N-terminal tryptophan over internal and C-terminal sequence isomers. Here, the binding of Q8 to aromatic peptides in the absence of methyl viologen was studied by isothermal titration calorimetry (ITC), (1)H NMR spectroscopy, and X-ray crystallography. The peptides studied were of sequence X-Gly-Gly, Gly-X-Gly, and Gly-Gly-X (X = Trp, Phe, Tyr, and His). Q8 selectively binds and dimerizes Trp-Gly-Gly (1) and Phe-Gly-Gly (4) with high affinity (ternary K = 10(9)-10(11) M(-)(2)); binding constants for the other 10 peptides were too small to be measured by ITC. Both peptides bound in a stepwise manner, and peptide 4 bound with positive cooperativity. Crystal structures of Q8.1 and Q8.4(2) reveal the basis for selective recognition as simultaneous inclusion of the hydrophobic aromatic side chain into the cavity of Q8 and chelation of the proximal N-terminal ammonium group by carbonyl groups of Q8. The peptide sequence selectivity and positively cooperative dimerization reported here are, to the best of our knowledge, unprecedented for synthetic hosts in aqueous solution. Specific peptide recognition and dimerization by synthetic hosts such as Q8 should be important in the study of dimer-mediated biochemical processes and for the separation of peptides and proteins.     

Proline hydroxylation alters the electrophoretic mobility of pulmonary surfactant-associated protein A

Studies from several laboratories involving amino acid analysis and sequencing of the Mr 35,000 pulmonary surfactant-associated proteins (SP-A) have detected hydroxyproline residues. These residues are present in a region with a collagen-like sequence that has been revealed by direct amino acid sequencing and from the deduced amino acid sequence of the cDNA clones coding for SP-A. We treated human lung tissue with tunicamycin to block N-glycosylation and with 2,2-dipyridyl to inhibit the hydroxylation of proline residues. The SP-A synthesized under these conditions showed a shift in apparent molecular weight to 27,000 and 29,000 compared to 29,000 and 31,000 for SP-A synthesized in the presence of tunicamycin alone. Dipyridyl treatment alone caused an alteration in electrophoretic mobility similar to that seen with tunicamycin, although this was more difficult to evaluate since changes in molecular weight due to glycosylation occurred under these conditions. These results indicate that proline hydroxylation in the collagen-like portion of SP-A decreases its electrophoretic mobility.     

Study of human neutrophil peptides in saliva by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry is used to rapidly characterize the human neutrophil peptides – HNP 1, 2, and 3 – in saliva. The saliva excreted from the parotid and sublingual/submandibular glands of 70 individuals were collected and examined using MALDI‐TOF. The MALDI approach requires no sample pretreatment other than mixing the saliva‐absorbing material with the matrix and drying under ambient conditions. Tissue paper was the best material for collecting the saliva samples because of its strong texture and high absorbance, and sinapinic acid was the best MALDI matrix for the analysis of the HNPs. HNPs were detected in almost all the samples collected from the parotid glands, with no obvious differences among age or gender. In contrast, the distribution of the HNPs in the samples collected from the sublingual/submandibular glands was age‐dependent: no HNPs were detected for those collected from individuals younger than 30, but the HNPs were present in all of the samples collected from those older than 60 years. The increased probability of detecting saliva HNPs with age suggests that HNPs may function as a biomarker for aging. Copyright © 2009 John Wiley & Sons, Ltd.     

Peptidomic analysis of human acquired enamel pellicle

Human acquired enamel pellicle is the result of a selective interaction of salivary proteins and peptides with the tooth surface. In the present work, the characterization of the peptides as well as the type of interactions established with the enamel surface was performed. Peptides from in vivo bovine enamel implants in the human oral cavity were sequentially extracted using guanidine and trifluoroacetic acid solutions and the fractions obtained were analysed by LC-MS and LC-MS/MS. Based on the LC-MS data, six phosphorylated peptides were identified in an intact form, strongly adsorbed to the enamel surface. Data from the LC-MS/MS analyses allowed us to identified 30 fragment peptides non-covalently bonded to enamel [basic proline-rich proteins, histatins (1 and 3) and acidic proline-rich protein classes]. The tandem mass spectrometry experiments showed the existence of a pattern of amide bond cleavage for the different identified peptide classes suggesting a selective proteolytic activity. For histatins, a predominance of cleavage at Arg, Lys and His residues was observed, while for basic proline-rich proteins, cleavage at Arg and Pro residues prevailed. In the case of acidic proline-rich proteins, a clearly predominance of cleavage of the Gln-Gly amide bond was evident.     

Exogenous pulmonary surfactant: A review focused on adjunctive therapy for severe acute respiratory syndrome coronavirus 2 including SP-A and SP-D as added clinical marker

Type I and type II pneumocytes are two forms of epithelial cells found lining the alveoli in the lungs. Type II pneumocytes exclusively secrete ‘pulmonary surfactants,’ a lipoprotein complex made up of 90% lipids (mainly phospholipids) and 10% surfactant proteins (SP-A, SP-B, SP-C, and SP-D). Respiratory diseases such as influenza, severe acute respiratory syndrome coronavirus infection, and severe acute respiratory syndrome coronavirus 2 infection are reported to preferentially attack type II pneumocytes of the lungs. After viral invasion, consequent viral propagation and destruction of type II pneumocytes causes altered surfactant production, resulting in dyspnea and acute respiratory distress syndrome in patients with coronavirus disease 2019. Exogenous animal-derived or synthetic pulmonary surfactant therapy has already shown immense success in the treatment of neonatal respiratory distress syndrome and has the potential to contribute efficiently toward repair of damaged alveoli and preventing severe acute respiratory syndrome coronavirus 2–associated respiratory failure. Furthermore, early detection of surfactant collectins (SP-A and SP-D) in the circulatory system can be a significant clinical marker for disease prognosis in the near future.     

Studies on the exchange of early pellicle proteins by mucin and whole saliva

Adsorption of small pellicle proteins statherin or proline-rich protein 1 (PRP1), respectively, and subsequent adsorption of human whole saliva (HWS) or salivary mucin MUC5B, respectively, was studied using ellipsometry and total internal reflectance fluorescence. Differences in elution (using sodium dodecyl sulphate (SDS) solutions) between mixed and single protein films were also investigated. On both hydrophilic and hydrophobized surfaces HWS and MUC5B were found to adsorb to pre-adsorbed layers of statherin and PRP1, respectively. Statherin adsorption on both substrate types showed no or minor exchange by HWS or MUC5B and no change in SDS elution between mixed and single protein films. Small amounts of PRP1 were exchanged by HWS on both surface types and the SDS elutable fractions were similar or larger for mixed films compared to single protein films. PRP1 and MUC5B in sequence showed minor exchange of PRP1 on hydrophilic surfaces, while no exchange could be established on hydrophobized substrates. SDS elutable fractions decreased for PRP1 and MUC5B mixed films compared to single protein films. In conclusion, minor amounts of statherin and PRP1 are exchanged during the time course of the experiments, which indicates that these proteins may to a large extent remain incorporated in the pellicle.     

Evaluation of carrier ampholyte-based capillary electrophoresis for separation of peptides and peptide mimetics

Carrier ampholyte-based capillary electrophoresis (CABCE) has recently been introduced as an alternative to CE (CZE) in the classical buffers. In this study, isoelectric BGEs were obtained by fractionation of Servalyt pH 4-9 carrier ampholytes to cuts of typical width of 0.2 pH unit. CABCE feasibility was examined on a series of insect oostatic peptides, i.e. proline-rich di- to decapeptides, and phosphinic pseudopeptides--tetrapeptide mimetics synthesized as a mixture of four diastereomers having the -P(O)(OH)-CH(2)- moiety embedded into the peptide backbone. With identical selectivity, the separation efficiency of CABCE proved to be as good as classical CE for the insect oostatic peptides and better for diastereomers of the phosphinic pseudopeptides. In addition, despite the numerous species present in the narrow pH cuts of carrier ampholytes, CABCE seems to be free of system zones that could hamper the analysis. Peak symmetry was good for moderately to low mobile peptides, whereas some peak distortion due to electromigration dispersion, was observed for short peptides of rather high mobility.     

Conformational changes in salivary proline-rich protein 1 upon adsorption to calcium phosphate crystals

Conformational analyses of PRP1, a proline-rich acidic salivary protein and major component of the acquired enamel pellicle, have been carried out in solution and upon binding to two enamel prototypes, hydroxyapatite (HA) and carbonated hydroxyapatite (CHA), using Fourier transform infrared spectroscopy (FTIR) in attenuated total reflection (ATR) mode. We have shown for the first time that, in solution, large portions of PRP1 adopt the hydrated polyproline type II (PPII) helical structure in addition to the random coil structure, with the maximum absorbance of the amide I band around 1620 cm(-1). Upon binding to HA or CHA, the protein undergoes significant conformational changes, loosing a considerable portion of hydrated PPII and random coil domains with a shift in the maximum absorbance to 1666 cm(-1), indicating that a large fraction of the protein is composed of beta turns. A small fraction of PPII in a calcium-bound or anhydrous form (approximately 1642 cm(-1)) was also observed in the HA- and CHA-bound proteins, which could play a role in protein-mineral interactions. The conformational changes in PRP1 adsorbed on CHA and HA were similar in nature; however, these changes were greater in the protein bound to HA. Interestingly, these results are in agreement with protein adsorption data that show that less protein is adsorbed onto CHA than onto HA. Our results demonstrate that binding to apatitic mineral surfaces leads to major conformational changes in PRP1, which might reflect the expulsion of water and the formation of protein-mineral and/or protein-protein interactions in the adsorbed layer.     

Simple, helical peptoid analogs of lung surfactant protein B

The helical, amphipathic surfactant protein, SP-B, is a critical element of pulmonary surfactant and hence is an important therapeutic molecule. However, it is difficult to isolate from natural sources in high purity. We have created and studied three different, nonnatural analogs of a bioactive SP-B fragment (SP-B(1-25)), using oligo-N-substituted glycines (peptoids) with simple, repetitive sequences designed to favor the formation of amphiphilic helices. For comparison, a peptide with a similar repetitive sequence previously shown to be a good SP mimic was also studied, along with SP-B(1-25) itself. Surface pressure-area isotherms, surfactant film phase morphology, and dynamic adsorption behavior all indicate that the peptoids are promising mimics of SP-B(1-25). The extent of biomimicry appears to correlate with peptoid helicity and lipophilicity. These biostable oligomers could serve in a synthetic surfactant replacement to treat respiratory distress syndrome.     

An engineered third electrostatic constriction of aerolysin to manipulate heterogeneously charged peptide transport

Reading the primary sequence directly using nanopores remains challenging due to the complex building blocks of 20 proteinogenic amino acids and the corresponding sophisticated structures. Compared to the uniformly negatively charged polynucleotides, biological nanopores hardly provide effective ionic current responses to all heterogeneously charged peptides under nearly physiological pH conditions. Herein, we precisely design a N226Q/S228K mutant aerolysin which creates a new electrostatic constriction named R3 in-between two natural sensing regions for controlling the capture and translocation of heterogeneously charged peptides. At nearly physiological pH, the decoration of positive charges at this constriction gives a large velocity of electroosmotic flow (EOF), leading to a maximum 8-fold increase in frequency for the heterogeneously charged peptides with the net charge from +1 to −3. Even the duration time of the negatively charged peptide Aβ35-25D4 in N226Q/S228K AeL also rises from 0.07 ± 0.01 ms to 0.63 ± 0.01 ms after introducing the third electrostatic constriction. Therefore, the N226Q/S228K aerolysin nanopore with three electrostatic constrictions realizes the dual goals of both capturing and decelerating heterogeneously charged peptides without labelling, even for the folded peptides.

An engineered aerolysin nanopore captures all types of peptides despite the charges and folded structure, which facilitate the achievement of nanopore protein sequencing.     

Analysis of salivary peptides using HPLC-electrospray mass spectrometry

Salivary peptides are involved in a wide range of functions constituting the first line of defence of oral cavity and precursors of dental pellicle formation. The presence of mucins in saliva makes difficult the analysis of the proteic content. This is due mainly to aggregation phenomenon between mucins and other high molecular weight glycoproteins and salivary proteins. Considering the importance of salivary peptides in biological functions, we have evaluated the influence of four different extraction methodologies on the separation and identification of these proteins by HPLC-MS. Based on their molecular weight, we identified a total of 22 peptides when extraction was performed using a solution of guanidine (6 m), compared with 14 peptides identified when saliva is acidified with TFA, which is an often used procedure. Our results also show the presence of mucin bind peptides, which include statherin, PRP1, PRP3, Histatin 1 and Histatin 5.     

<Emphasis Type="SmallCaps">d</Emphasis>-Amino Acids in Animal Peptides

Summary. Secreted peptides from diverse sources have been found to contain a d-amino acid. From the sequence of cloned mRNAs coding for the precursors of such peptides it could be deduced that in all cases tested so far the d-amino acid in the final product is derived from the corresponding l-amino acid present in the primary product of translation. Enzymes catalyzing such an l- to d-isomerization in peptide linkage have been isolated from the venom of a spider and the skin secretions of frogs. Even though these are completely different proteins, the reaction mechanism is the same, namely a de-protonation/re-protonation of the α-carbon of an amino acid with concomitant inversion of the chirality. Sequences potentially coding for homologues of the frog enzyme are present in the genome of different vertebrate species.     

Separation of enantiomers on anion exchangers modified with heparin in liquid chromatography

Hepatitis B virus surface antigen (HBsAg) particles were efficiently adsorbed (retained) on a Sulfate-cellulose (S-C) bead column, and then desorbed with sodium chloride solutions (0.5-3.0 M). The HBsAg particles were not efficiently retained onto either sulfopropyl-agarose (SP-A) or quaternary amine-agarose (Q-A) at pH 4.5, 6 and 8. The size-exclusion curve showed that proteins of molecular mass higher than ca. 20,000 cannot penetrate into the pores of S-C beads. The dynamic binding capacity (DBC) values of lysozyme (ca. 7 mg/ml-gel) and of gamma-globulin (ca. 3 mg/ml gel) for S-C did not depend on the flow velocity while the DBC of gamma-globulin for SP-A decreased sharply with an increase in flow velocity. These results indicated that very large molecules are adsorbed only onto the surface of S-C, which resulted in fast adsorption-desorption rates although the equilibrium adsorption capacity is lower than conventional porous gel beads. Because of the rapid adsorption rate, the DBC values of gamma-globulin for S-C at high flow-rate regions are similar to those for SP-A. Bovine serum albumin was not adsorbed onto S-C. As this can not be explained by a simple electrostatic interaction mechanism, molecular recognition of S-C might be different from the agarose-based ion-exchange beads.     

On the structural diversity of sialoliths.

Sialoliths from parotid and submaxillar glands have been characterized. Fractured and polished surfaces revealed an intrinsic structural diversity across the calculi sections. In general, the calculi presented highly mineralized amorphous-looking cores surrounded by concentric alternating mineralized and organic layers. The thickness of these layers decreased from the outer regions toward the center of the sialolith, illustrating a sequence of growth stages. Nevertheless, a significant variability could be detected among the specimens. In some cases, the calculi displayed multiple cores and lacked concentric laminated structures. In other instances, the specimens exhibited extensive regions of globular structures. In these cases, the globule diameter decreased across the radius toward the center of the sialoliths, and the globular structures tended to reorganize, forming bright and dark laminated layers surrounding the core. The participation of globular structures in the layer formation process points to morphogenetic mechanisms not previously described.     

pH dependence of the crystal violet adsorption isotherm at the silica-water interface

The pH-dependent adsorption isotherms for the charged chromophore crystal violet, CV(+), have been measured with three different bases by a free-running cavity implementation of evanescent wave cavity ring-down spectroscopy. The ratio of the maximal absorbance measurements at pH 5.10 and 9.05 is consistent with a Q2:Q3 silanol site ratio of 72.8:27.2. The adsorption isotherms have been interpreted in terms a cooperative binding adsorption allowing more than one ionic species to bind to each silanol group. The surface concentration is consistent with a silanol charge density of 1.92 +/- 0.55 nm(-2) and a total neutralized interface layer structure extending 9 nm from the surface. Binding constants and stoichiometric coefficients are derived for CV(+) to both the Q2 and Q3 sites. A variation of the adsorption isotherm with base is observed so that the isotherm at pH 9.05 adjusted with ammonium hydroxide sets up a competitive acid-base equilibrium with the SiOH groups with only 49% of the surface silanol sites dissociated. The implications for functionalized surfaces in chromatography are discussed.     

Expanded polyglutamine-binding peptoid as a novel therapeutic agent for treatment of Huntington's disease

Polyglutamine(polyQ)-expanded proteins are potential therapeutic targets for the treatment of polyQ expansion disorders such as Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3). Here, we used an amino-terminal fragment of a mutant Huntingtin protein (Htt-N-82Q) as bait in an unbiased screen of a 60,000 peptoid library. Peptoid HQP09 was selected from the isolated hits and confirmed as a specific ligand of Htt-N-82Q and Atxn3-77Q mutant proteins in biochemical experiments. We identified three critical residues in the HQP09 sequence that are important for its activity and generated a minimal?derivative, HQP09_9, which maintains the specific polyQ-binding activity. We demonstrated that HQP09 and HQP09_9 inhibited aggregation of Htt-N-53Q in?vitro and exerted Ca(2+)-stabilizing and neuroprotective effects in experiments with primary striatal neuronal cultures derived from HD mice. We further demonstrated that intracerebroventricular delivery of HQP09 to an HD mouse model resulted in reduced accumulation of mutant Huntingtin aggregates and improved motor behavioral outcomes. These results suggest that HQP09 and similar peptoids hold promise as novel therapeutics for developing treatments for HD, SCA3, and other polyglutamine expansion disorders.     

Radioreceptor Assay of Opioid Peptides in Selected Canine Brain Regions

Abstract

A radioreceptor assay using the opioid delta receptor-preferring ligand D- ²ala, D- ⁵leu leucine enkephalin (³H-DADL) and the broader-specificity ligand ³H-etorphine was used to measure five HPLC-purified neuropeptide fractions derived from the peptide-rich fraction of tissue homogenates of nine anatomical regions of the canine brain. The receptoractive peptides studied were methionine enkephalin, alpha-neo-endorphin, dynorphin 1-8, methionine enkephalin-Arg-Phe, and leucine enkephalin. These peptides derive from two larger precursors: proenkephalin A, which contains methionine enkephalin, leucine enkephalin, methionine enkephalin-Arg-Phe; and proenkephalin B, which contains alpha-neo-endorphin and dynorphin 1-8. Receptoractive peptides were measured in the peptide-rich fraction derived from homogenates of canine hypothalamus, pituitary, caudate nucleus, amygdala, hippocampus, mid-brain, thalamus, pons-medulla, and cortex.     

氨基酸描述子VHSEH用于多肽定量序效建模研究

从20 种天然氨基酸的171个物化性质出发, 按照疏水、立体和电性特征及氢键贡献将其分类后, 分别进行主成分分析, 得到一个新描述子VHSEH(Principal component score vector of hydrophobic, steric, electronic properties and, hydrogen bonds contributions). 对后叶催产素的结构进行了表征, 并以偏最小二乘法及D-优化划分样本建立了PLS定量序效关系模型, 得到复相关系数R2分别为 0.958 和 0.957, Q2分别为0.903和0.845, 约高于VHSE描述子模型值; 对抗菌肽进行了结构表征, 建立了PLS和OSC-PLS模型, 其R2分别为0.84和 0.995, Q2分别为0.546和0.926, 较SZOTT描述子结果好; 对58 个血管紧张素转化酶抑制剂进行QSAM研究, 得到R2, Q2及RMS分别为0.877, 0.838和0.361. 研究结果表明, VHSEH 描述子信息量大, 物化意义明确, 结果更易解释.