GRable Version 1.0: A Software Tool for Site-Specific Glycoform Analysis With Improved MS1-Based Glycopeptide Detection With Parallel Clustering and Confidence Evaluation With MS2 Information.

High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here, we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points of the algorithms are "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large-scale and in-depth. Therefore, this software will help us analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can be freely run online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable).

Combinatorial Approach with Mass Spectrometry and Lectin Microarray Dissected Site-Specific Glycostem and Glycoleaf Features of the Virion-Derived Spike Protein of Ancestral and γ Variant SARS-CoV-2 Strains.

The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (γ-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the γ-variant S protein; however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.

Near-infrared radiation causes sebaceous gland enlargement along with an ROS-dependent augmentation of epidermal growth factor receptor expression in hamsters.

As near-infrared radiation (NIR), which is a composition of sunlight with an 780-1400 nm wavelength, is associated with skin aging such as wrinkles and slacks, the biological actions of NIR with high dermal penetration remains unclear. In the present study, we found that NIR irradiation (40 J/cm2 ) at different levels of irradiance (95-190 mW/cm2 ) using a laboratory device with a xenon flash lamp (780-1700 nm) caused sebaceous gland enlargement concomitantly with skin thickening in the auricle skin of hamsters. The sebaceous gland enlargement resulted from the proliferation of sebocytes due to an increase in the number of proliferating cell nuclear antigen (PCNA)- and lamin B1-positive cells in vivo. In addition, NIR irradiation transcriptionally augmented the production of epidermal growth factor receptor (EGFR) accompanied with an increase in the reactive oxygen species (ROS) level in hamster sebocytes in vitro. Furthermore, the administration of hydrogen peroxide increased the level of EGFR mRNA in the sebocytes. Therefore, these results provide novel evidence that NIR irradiation causes the hyperplasia of sebaceous glands in hamsters by mechanisms in which EGFR production is transcriptionally augmented through ROS-dependent pathways in sebocytes.

Molecular mechanisms of cyclic phosphatidic acid-induced lymphangiogenic actions in vitro.

The lymphatic system plays important roles in various physiological and pathological phenomena. As a bioactive phospholipid, lysophosphatidic acid (LPA) has been reported to function as a lymphangiogenic factor as well as some growth factors, yet the involvement of phospholipids including LPA and its derivatives in lymphangiogenesis is not fully understood. In the present study, we have developed an in-vitro lymphangiogenesis model (termed a collagen sandwich model) by utilizing type-I collagen, which exists around the lymphatic endothelial cells of lymphatic capillaries in vivo. The collagen sandwich model has revealed that cyclic phosphatidic acid (cPA), and not LPA, augmented the tube formation of human dermal lymphatic endothelial cells (HDLECs). Both cPA and LPA increased the migration of HDLECs cultured on the collagen. As the gene expression of LPA receptor 6 (LPA6) was predominantly expressed in HDLECs, a siRNA experiment against LPA6 attenuated the cPA-mediated tube formation. A synthetic LPA1/3 inhibitor, Ki16425, suppressed the cPA-augmented tube formation and migration of the HDLECs, and the LPA-induced migration. The activity of Rho-associated protein kinase (ROCK) located at the downstream of the LPA receptors was augmented in both the cPA- and LPA-treated cells. A potent ROCK inhibitor, Y-27632, suppressed the cPA-dependent tube formation but not the migration of the HDLECs. Furthermore, cPA, but not LPA, augmented the gene expression of VE-cadherin and ß-catenin in the HDLECs. These results provide novel evidence that cPA facilitates the capillary-like morphogenesis and the migration of HDLECs through LPA6/ROCK and LPA1/3 signaling pathways in concomitance with the augmentation of VE-cadherin and ß-catenin expression. Thus, cPA is likely to be a potent lymphangiogenic factor for the initial lymphatics adjacent to type I collagen under physiological conditions.

Effect of CYP3A5*3 genetic variant on the metabolism of direct-acting antivirals in vitro: a different effect on asunaprevir versus daclatasvir and beclabuvir.

Direct-acting antivirals, asunaprevir (ASV), daclatasvir (DCV), and beclabuvir (BCV) are known to be mainly metabolized by CYP3A enzymes; however, the differences in the detailed metabolic activities of CYP3A4 and CYP3A5 on these drugs are not well clarified. The aim of the present study was to elucidate the relative contributions of CYP3A4 and CYP3A5 to the metabolism of ASV, DCV, and BCV, as well as the effect of CYP3A5*3 genetic variant in vitro. The amount of each drug and their major metabolites were determined using LC-MS/MS. Recombinant CYP3As and CYP3A5*3-genotyped human liver microsomes (CYP3A5 expressers or non-expressers) were used for the determination of their metabolic activities. The contribution of CYP3A5 to ASV metabolism was considerable compared to that of CYP3A4. Consistently, ASV metabolic activity in CYP3A5 expressers was higher than those in CYP3A5 non-expresser. Moreover, CYP3A5 expression level was significantly correlated with ASV metabolism. In contrast, these observations were not found in DCV and BCV metabolism. To our knowledge, this is the first study to directly demonstrate the effect of CYP3A5*3 genetic variants on the metabolism of ASV. The findings of the present study may provide basic information on ASV, DCV, and BCV metabolisms.

Minor contribution of CYP3A5 to the metabolism of hepatitis C protease inhibitor paritaprevir in vitro.

Paritaprevir (PTV) is a non-structural protein 3/4A protease inhibitor developed for the treatment of hepatitis C disease as a fixed dose combination of ombitasvir (OBV) and ritonavir (RTV) with or without dasabuvir. The aim of this study was to evaluate the effects of cytochrome P450 (CYP) 3A5 on in vitro PTV metabolism using human recombinant CYP3A4, CYP3A5 (rCYP3A4, rCYP3A5) and human liver microsomes (HLMs) genotyped as either CYP3A5*1/*1, CYP3A5*1/*3 or CYP3A5*3/*3. The intrinsic clearance (CLint, Vmax/Km) for the production of a metabolite from PTV in rCYP3A4 was 1.5 times higher than that in rCYP3A5. The PTV metabolism in CYP3A5*1/*1 and CYP3A5*1/*3 HLMs expressing CYP3A5 was comparable to that in CYP3A5*3/*3 HLMs, which lack CYP3A5. CYP3A4 expression level was significantly correlated with PTV disappearance rate and metabolite formation. In contrast, there was no such correlation found for CYP3A5 expression level. This study represents that the major CYP isoform involved in PTV metabolism is CYP3A4, with CYP3A5 having a minor role in PTV metabolism. The findings of the present study may provide foundational information on PTV metabolism, and may further support dosing practices in HCV-infected patients prescribed PTV-based therapy.

D-Amino acids in protein: The mirror of life as a molecular index of aging.

Proteins are composed exclusively of l-amino acids. Among elderly individuals, however, d-aspartic acid (d-Asp) residues have been found in eye lens and brain, as well as in other tissues. The presence of d-Asp may change the higher-order structure of a protein, which in turn may have a role in age-related disorders such as cataract and Alzheimer's disease. d-Asp results from the spontaneous racemization of Asp residues in susceptible proteins. During aging, natural lα-Asp residues in proteins are non-enzymatically isomerized via a succinimidyl intermediate to l-ß-, d-α- and d-ß-isomers. This isomerization does not happen uniformly, but instead occurs at specific residues that are susceptible to isomerization due to their sequence or structural context. Thus, it is necessary to establish the nature of each individual Asp residue in susceptible proteins. Recently, a new method based on LC-MS/MS for the analysis of Asp isomerization at specific protein sites has been described. In this review, we first show that the homochirality of amino acids in proteins is not guaranteed throughout life. We then describe the development of a new method for protein-bound d-amino acid analysis, and discuss the negative influence that d-Asp has on protein structure and function.

Alpha B- and ßA3-crystallins containing d-aspartic acids exist in a monomeric state.

Crystallin stability and subunit-subunit interaction are essential for eye lens transparency. There are three types of crystallins in lens, designated as α-, ß-, and γ-crystallins. Alpha-crystallin is a hetero-polymer of about 800kDa, consisting of 35-40 subunits of two different αA- and αB-subunits, each of 20kDa. The ß/γ-crystallin superfamily comprises oligomeric ß-crystallin (2-6 subunits) and monomeric γ-crystallin. Since lens proteins have very long half-lives, they undergo numerous post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation, which may decrease crystallin solubility and ultimately cause cataract formation. Racemization and isomerization of aspartyl (Asp) residues have been detected only in polymeric α- and oligomeric ß-crystallin, while the situation in monomeric γ-crystallin has not been studied. Here, we investigated the racemization and isomerization of Asp in the γ-crystallin fraction of elderly donors. The results show that Asp residues of γS-, γD- and γC-crystallins were not racemized and isomerized. However, strikingly, we found that a portion of αB-crystallin and ßA3-crystallin moved to the lower molecular weight fraction which is the same size of γ-crystallin. In those fractions, Asp-96 of αB-crystallin and Asp-37 of ßA3-crystallin were highly inverted, which do not occur in the native lens higher molecular weight fraction. Our results indicate the possibility that the inversion of Asp residues may induce dissociation of αB- and ßA3-crystallins from the polymeric and oligomeric states. This is the first report that stereoinversion of amino acids disturbs lens protein assembly in aged human lens.

Rapid survey of four Asp isomers in disease-related proteins by LC-MS combined with commercial enzymes.

Until relatively recently, it was considered that D-amino acids were excluded from living systems except for the cell wall of microorganisms. However, D-aspartate residues have now been detected in long-lived proteins from various tissues of elderly humans. Formation of D-aspartate in proteins induces aggregation and loss of function, leading to age-related disorders such as cataracts and Alzheimer disease. A recent study used LC-MS to analyze isomers of Asp residues in proteins precisely without complex purification of the proteins. However, to identify the four Asp isomers (Lα, Lß, Dß, and Dα) on the chromatogram, it was necessary to synthesize reference peptides containing the four different Asp isomers as standards. Here, we describe a method for rapidly and comprehensively identifying Asp isomers in proteins using a combination of LC-MS and commercial enzymes without synthesizing reference peptides. The protein sample is treated with trypsin, trypsin plus Asp-N, trypsin plus PIMT, trypsin plus paenidase, and the resulting peptides are applied to LC-MS. Because Asp-N hydrolyzes peptide bonds on the N-terminus of only Lα-Asp residues, it differentiates peptides containing Lα-Asp from those containing the other three isomers. Similarly, PIMT recognizes only peptides containing Lß-Asp residues, and paenidase internally cleaves the C-terminus of Dα-Asp residues. This approach was successfully applied to the analysis of all tryptic peptides in aged lens. The comprehensive quantitative data of Asp isomer formation in age-related proteins obtained via this method might be used as biomarkers of age-related disease.

Real-time heterogeneous protein-protein interaction between αA-crystallin N-terminal mutants and αB-crystallin using quartz crystal microbalance (QCM).

The lens transparency depends on higher concentration of lens proteins and their interactions. α-Crystallin is one of the predominant lens proteins, responsible for proper structural and functional architecture of the lens microenvironment, and any alteration of which results in cataract formation. The R12C, R21L, R49C and R54C are the most significant and prevalent αA-crystallin congenital cataract-causing mutants worldwide. Protein-protein interaction, crucial for lens proper structure and function, was posited to be lost due to point mutation and the elucidation of which could shed light on the molecular basis of cataract. In this conjuncture, we report quartz crystal microbalance (QCM) as a warranted technique for real-time analysis of protein-protein interaction between the N-terminal mutants of αA-crystallin and αB-crystallin. The biophysical characteristics of the mutated proteins were determined by size-exclusion HPLC, far-UV circular dichroism and fluorescence studies. Far-UV circular dichroism spectral analysis displayed slight modifications in ß-sheet of R54C mutant. Altered intrinsic tryptophan fluorescence and decreased bis-ANS fluorescence were observed in all the N-terminal mutations revealing the tertiary structural changes and decreased exposure of surface hydrophobicity. An emphatic fall in the chaperone activity was observed in the N-terminal mutants, R12C, R21L and R54C. QCM analysis revealed the occurrence of strong heterogeneous interaction between αA-crystallin and αB-crystallin. Nevertheless, decreased interactions were observed with the N-terminal mutants. In summary, the present study concludes that the loss of interactions between αA-crystallin N-terminal mutants and αB-crystallin signifies quaternary structural alterations due to mutation in the arginine residues.

Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

PURPOSE: To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays. METHODS: Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay. RESULTS: αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed ß-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant. CONCLUSIONS: Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

isoAsp-Quest: Workflow development for isoAsp identification using database searches.

A recent study reported that isomerization of aspartyl residues (Asp) occurs in various tissues and proteins in vivo. For a comprehensive analysis of post-translational modifications, the MS-based proteomic approach is a straightforward method; however, the isomerization of Asp does not alter its molecular weight. Therefore, a unique method is required to analyze Asp isomers using mass spectrometry. Herein, we present a novel strategy, isoAsp-Quest, which is a database search-oriented isoAsp identification method. isoAsp is specifically converted to 18O-labeled Lα-Asp by the enzymatic reaction of protein L-isoaspartyl-O-methyltransferase (PIMT) in 18O water with a mass shift of 2 Da, which, in principle, enables us to distinguish Asp isomers. However, in practice, a labeled Lα-Asp signal overlaps with that of endogenous Lα-Asp, making detection challenging. Therefore, degradation of the endogenous Lα-Asp peptide by AspN and subsequent removal of AspN were performed prior to the PIMT reaction. This strategy was applied to bovine lens α-crystallin. Consequently, several Asp isomerization sites, consistent with human αA-crystallin, were identified in bovine αA-crystallin, indicating that this strategy is also effective for biological proteins. Therefore, isoAsp-Quest enables the analysis of Lß-Asp in a straightforward and rapid workflow, which may be useful for the quality control of protein products and biomarker discovery.

Glycosylation of recombinant adeno-associated virus serotype 6.

Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galß1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%-1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

A rapid, comprehensive liquid chromatography-mass spectrometry (LC-MS)-based survey of the Asp isomers in crystallins from human cataract lenses.

Cataracts are caused by clouding of the eye lens and may lead to partial or total loss of vision. The mechanism of cataract development, however, is not well understood. It is thought that abnormal aggregates of lens proteins form with age, causing loss of lens clarity and development of the cataract. Lens proteins are composed of soluble α-, ß-, and γ-crystallins, and as long lived proteins, they undergo post-translational modifications including isomerization, deamidation, and oxidation, which induce insolubilization, aggregation, and loss of function that may lead to cataracts. Therefore, analysis of post-translational modifications of individual amino acid residues in proteins is important. However, detection of the optical isomers of amino acids formed in these proteins is difficult because optical resolution is only achieved using complex methodology. In this study, we describe a new method for the analysis of isomerization of individual Asp residues in proteins using LC-MS and the corresponding synthetic peptides containing the Asp isomers. This makes it possible to analyze isomers of Asp residues in proteins precisely and quickly. We demonstrate that Asp-58, -76, -84, and -151 of αA-crystallin and Asp-62 and -96 of αB-crystallin are highly converted to lß-, dß-, and dα-isomers. The amount of isomerization of Asp is greater in the insoluble fraction at all Asp sites in lens proteins, therefore indicating that isomerization of these Asp residues affects the higher order structure of the proteins and contributes to the increase in aggregation, insolubilization, and disruption of function of proteins in the lens, leading to the cataract.

An increase in normetanephrine in hair follicles of acne lesions through the sympatho-adrenal medullary system in acne patients with anxiety.

Acne vulgaris, a chronic inflammatory skin disease, has been associated with not only sebaceous gland dysfunction but also various endogenous and exogenous stresses. Since sebaceous glands are under neuroendocrine control, including the hypothalamic-pituitary-adrenal axis and neuro-autocrine mechanisms, it remains unclear how psychological stress relates to the pathogenesis of acne. In this study, we investigated the relationship between psychological stress and catecholamine in acne lesions from 18 patients with mild or moderate acne. The State-Trait Anxiety Inventory (STAI) revealed that all patients were anxious, with six having low anxiety and 12 high anxiety. Salivary α-amylase activity (sAA), which is regulated by the sympatho-adrenal medullary (SAM) system, positively correlated with the STAI State Anxiety scores (STAI-S) and was significantly detectable in acne patients with high rather than low anxiety. In addition, the level of normetanephrine, but not metanephrine, both of which are catecholamine metabolites, in hair follicles of acne lesions also positively correlated with the STAI-S. Furthermore, the normetanephrine level was higher in patients with high rather than low anxiety, whereas there was no change in metanephrine in the hair follicles of the acne lesions. Moreover, neither the sAA nor metanephrine and normetanephrine in the acne lesions was related to acne severity in the patients. Thus, these results provide novel evidence that a SAM system-associated increase of normetanephrine level in hair follicles is involved in the acne pathology of patients with anxiety.

D-Alanine Is Reduced by Ocular Hypertension in the Rat Retina.

Purpose: The purpose of this study was to investigate the physiological changes of amino acids in the rat retina caused by ocular hypertension.Methods: Adult Wistar rats were used as an experimental model of ocular hypertension. Retinas were hydrolyzed with HCl at 108°C to isolate amino acids. Residual amino acids were measured by reverse-phase high-performance liquid chromatography and the total volume of residual amino acids and the ratio of D- and L-amino acids were analyzed. Free D- and L-alanine levels were also measured using two-dimensional HPLC.Results: The amount of retinal alanine decreased in ocular hypertension compared with the control (p < .05, Student's t-test); the amounts of other amino acids did not differ between the two conditions.The D/L ratio of alanine was higher than that of other amino acids. Ocular hypertension reduced the D/L ratio of retinal alanine, while that of other amino acids was unchanged. Ocular hypertension increased the D/L ratio of free alanine.Conclusions: Ocular hypertension reduced the D/L ratio of retinal alanine, presumably due in large part to alanine peptides.

Isomeric Replacement of a Single Aspartic Acid Induces a Marked Change in Protein Function: The Example of Ribonuclease A.

lα-Aspartic acid (Asp) residues in proteins are nonenzymatically isomerized to abnormal lß-, dα-, and dß-Asp isomers under physiological conditions. Such an isomerization of Asp residues is considered to be a trigger of protein denaturation because it either elongates the main chain or induces a different orientation of the side chain within the protein structure or both. However, previous studies have found no direct evidence of the effects of Asp isomers on protein function. Therefore, the production of Asp-isomer-containing proteins is required to verify the effects of Asp isomerization. Here, we describe the production of an Asp-isomer-containing protein using the expressed protein ligation. As a model protein, bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5), which catalyzes the cleavage of phosphodiester bonds in RNA, was used. In this study, lα-Asp at position 121 in RNase A was replaced by lß-, dα-, and dß-Asp. The objective aspartic acid at position 121 is located near the active site and related to RNA cleavage. The RNase A with lα-Asp at position 121 showed a normal activity. By contrast, the catalytic activity of lß-, dα-, and dß-Asp-containing RNase A was markedly decreased. This study represents the first synthesis and analysis of a protein containing four different Asp isomers.

Mouse d-Amino-Acid Oxidase: Distribution and Physiological Substrates.

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties.

Proteomic study of retinal proteins associated with transcorneal electric stimulation in rats.

Background. To investigate how transcorneal electric stimulation (TES) affects the retina, by identifying those proteins up- and downregulated by transcorneal electric stimulation (TES) in the retina of rats. Methods. Adult Wistar rats received TES on the left eyes at different electrical currents while the right eyes received no treatment and served as controls. After TES, the eye was enucleated and the retina was isolated. The retinas were analyzed by proteomics. Results. Proteomics showed that twenty-five proteins were upregulated by TES. The identified proteins included cellular signaling proteins, proteins associated with neuronal transmission, metabolic proteins, immunological factors, and structural proteins. Conclusions. TES induced changes in expression of various functional proteins in the retina.