Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass

Left ventricular mass (LVM) and cardiac gene expression are complex traits regulated by factors both intrinsic and extrinsic to the heart. To dissect the major determinants of LVM, we combined expression quantitative trait locus1 and quantitative trait transcript (QTT) analyses of the cardiac transcriptome in the rat. Using these methods and in vitro functional assays, we identified osteoglycin (Ogn) as a major candidate regulator of rat LVM, with increased Ogn protein expression associated with elevated LVM. We also applied genome-wide QTT analysis to the human heart and observed that, out of 22,000 transcripts, OGN transcript abundance had the highest correlation with LVM. We further confirmed a role for Ogn in the in vivo regulation of LVM in Ogn knockout mice. Taken together, these data implicate Ogn as a key regulator of LVM in rats, mice and humans, and suggest that Ogn modifies the hypertrophic response to extrinsic factors such as hypertension and aortic stenosi

Proteomic Analysis of Potential Keratan Sulfate, Chondroitin Sulfate A, and Hyaluronic Acid Molecular Interactions

Published April 7, 2010 as a Recently Accepted Papers (RAPpers)Purpose: Corneal stroma extracellular matrix (ECM) glycosaminoglycans (GAGs) include keratan sulfate (KS), chondroitin sulfate A (CSA), and hyaluronic acid (HA). Embryonic corneal keratocytes and sensory nerve fibers grow and differentiate according to chemical cues they receive from their ECM. This study asks what proteins that might regulate keratocytes or corneal nerve growth cone immigration interact with corneal GAGs. Method: Biotinylated KS (bKS), CSA (bCSA), and HA (bHA) were prepared, and used in Invitrogen’s v4 protoarray to assess their interactions with 8268 proteins and a custom array of 85 extracellular epitopes of nerve growth-related proteins. Surface Plasmon Resonance (SPR) was performed with bKS and SLIT2, and their Ka, Kd, and KD values determined. Results: Highly sulfated KS interacted with 217 v4 proteins, including 75 kinases, several membrane or secreted proteins, many cytoskeletal proteins, and many nerve function proteins. CSA interacted with 24 v4 proteins, including 10 kinases and 2 cell surface proteins. HA interacted with 6 v4 proteins, including several ECM-related structural proteins. Of 85 ECM nerve-related epitopes, KS bound 40 proteins, including SLIT, 2 ROBOs, 9 EPHs, 8 Ephrins (EFNs), 8 semaphorins (SEMAs) and 2 nerve growth factor receptors. CSA bound 9 proteins, including ROBO2, 2 EPHs, 1 EFN, 2 SEMAs, and Netrin4. HA bound no ECM nerve-related epitopes. SPR confirmed that KS binds SLIT2 strongly. KS core protein mimecan/osteoglycin bound 15 v4 proteins. Conclusion: Corneal stromal GAGs bind, and thus could alter the availability or conformation of, many proteins that may influence keratocyte behavior and nerve growth cone behavior in the cornea

Detection and Quantification of Sulfated Disaccharides from Keratan Sulfate and Chondroitin/Dermatan Sulfate during Chick Corneal Development by ESI-MS/MS

PURPOSE. To identify and quantify changes in keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) sulfated disaccharides in the developing chick cornea using electrospray ionization tandem mass spectrometry (ESI-MS/MS). METHODS. Cryostat sections of fresh nonfixed corneas were obtained from White Leghorn embryonic day (E)8 to E20 chicks, and from 4-and 70-week-old chickens. Tissue sections on glass slides were incubated with selected glycosidase enzymes. Digest solutions were analyzed directly by ESI-MS/MS. RESULTS. The concentration of KS monosulfated disaccharide (MSD) Gal-␤-1,4-GlcNAc(6S) in E8 cornea equaled that at E20, declined to its lowest level by E10, increased to a second peak by E14, decreased to a second low by E18, peaked again by E20, and remained high in adult corneas. A similar concentration profile was observed for KS disulfated disaccharide (DSD) Gal(6S)-␤-1,4-GlcNAc(6S), and thus also for total sulfated KS disaccharides. The molar percent of DSD was higher than that of MSD from E8 to E18, equivalent at E20, and less than that of MSD in adult corneas. In contrast, total concentration of CS/DS ⌬di-4S plus ⌬di-6S decreases as development progresses and is lowest in adult corneas. Concentration and molar percent of ⌬di-6S is highest at E8, then decreases through development as the concentration and molar percent of ⌬di-4S increases from E8 and exceeds that of ⌬di-6S after E14. CONCLUSIONS. New, rapid, direct chemical analysis of extracellular matrix components obtained from sections from embryonic and adult chick corneas reveals heretofore undetected changes in sulfation characteristics of KS and CS/DS disaccharides during corneal development. (Invest Ophthalmol Vis Sci. 2005;46:1604 -1614) DOI:10.1167/iovs.04-1453 G lycosaminoglycans (GAGs) are negatively charged, highmolecular-weight polysaccharides classified on the basis of their structures into several groups: hyaluronan (HA), CS/ DS, KS, heparan sulfate (HS), and heparin (Hep). With the exception of KS, GAGs are composed of alternating residues of uronic acid and N-acetylhexosamine. Sulfate groups are attached to a limited number of hydroxyl or amino groups on GAGs and contribute greatly to their polyanionic properties. In cornea, KS, and CS/DS are the two major GAGs. 1 KS was first isolated from bovine cornea. 2 KS chains have been extracted from many tissues, and several KS-containing proteoglycans have been identified. KS chains have been classified according to their linkage to protein: KS-I for N-asparagine-linked chains from cornea and KS-II for O-serine-linked chains from skeletal tissues such as cartilage. A third type of KS, O-linked from mannose to serine or threonine, has been isolated from brain tissue. 14 Invasion of the stroma by more neural crest cells begins on E5. 14 Over the next several days, these invading stromal fibroblastic cells assume the stellate morphology of keratocytes and secrete the connective tissue that characterizes the secondary or adult stroma. Beginning at about E14, under the influence of thyroxine, the stroma starts to dehydrate and become thinner and more transparent, with full transparency being achieved just before hatching on E20 to E21. 12 Combining use of labeled precursors and HPLC techniques, KS and CS/DS sulfated disaccharides from GAGs labeled during explant culture have been examined during corneal development. With the development of ionization methods, electrospray ionization (ESI)-MS/MS techniques have become more attractive and prominent for the analysis of oligosaccharides, 18 because they provide high mass accuracy, structural information, and ability to quantify the fragments, 19 -23 and because they do not require labeling of the GAGs in explant culture before analysis. It has also been demonstrated that MS/MS techniques can be used for compositional analysis of purified chondroitin sulfate (CS) and heparan sulfate (HS) polymers that have been hydrolyzed by enzymes. 24 -27 More recently, the KS disaccharides Gal-␤-1,4-GlcNAc(6S) (monosulfated disaccharide; MSD) and Gal(6S)-␤-1,4-GlcNAc(6S) (disulfated disaccharide; DSD), released from bovine cornea, bovine nasal cartilage, mouse brain, and rat brain by enzymatic digestion and isolated by liquid chromatography, have been analyzed by turbo ion spray tandem mass spectrometry (LC-MS/MS). In this study, we use ESI-MS/MS techniques to directly identify and quantify KS and CS/DS sulfated disaccharides liberated from single frozen sections of developing chick cornea by enzymatic digestion, without purification of digestion products or chromatographic separation preceding mass spectral analysis. By such an approach, the proportions of KS and CS/DS sulfated disaccharides during chick corneal development were determined. MATERIALS AND METHODS Reagents KS Gal-␤-1,4-GlcNAc(6S) and Gal(6S)-␤-1,4-GlcNAc(6S) were kindly provided by Seikagaku Corporation (Tokyo, Japan). Disaccharides of CS/DS-⌬di-2S, which was also used as the internal standard for quantification of KS sulfated disaccharides, ⌬di-4S, ⌬di-6S, and chondroitinase ABC (from Proteus vulgaris, protease free, EC 4.2.2.4)-were purchased from Sigma-Aldrich (St. Louis, MO). ⌬UA-␤-1,4-GlcNS, which was used as the internal standard for quantification of CS/DS sulfated disaccharides, was purchased from EMD Biosciences, Inc. (San Diego, CA). Keratanase II (from Bacillus sp.) was purchased from Seikagaku America (Falmouth, MA). Ammonium acetate and ammonium sulfate were purchased from Sigma-Aldrich. Solvents used were of HPLC grade and were purchased from Fisher Scientific (Pittsburgh, PA). Centrifugal filter units (Ultra free-MC, 5000 NMWL; Millipore, Bedford, MA) were purchased from Fisher Scientific. Preparation of Standard Solutions All disaccharide standards were diluted in a series of 10 nmol/L, 1 nmol/L, 100 pmol/L, and 10 pmol/L in water, and aliquots were stored at Ϫ20°C. For qualitative analysis, 10 L of the 1 nmol/L solution of each standard was diluted by adding 70 L of MeOH, 5 L of 5 mM ammonium acetate buffer (pH 7.5), 5 L of 2 mM (NH 4 ) 2 SO 4 , and 10 L of water to make the solution 7:3 MeOH/H 2 O, containing 100 pmol/L of disaccharide standard sample. The addition of (NH 4 ) 2 SO 4 was helpful in suppressing sodiated adducts. 24 Preparation of Tissue Sections All animals were used in accordance with the ARVO Statement for the Use of Animals for Ophthalmic and Vision Research. Corneas were dissected from White Leghorn embryonic chicks of ages ranging from embryonic day (E)8 to E20, as well as from 4-and 70-week-old chickens. Fresh corneas were washed three times in saline solution and then embedded in optimal cutting temperature polymer (Sakura Finetek USA. Inc., Torrance, CA) in liquid nitrogen. Sections of 10-m thickness were cut on a cryostat (Hacker-Bright, Huntington, UK) at Ϫ24°C, collected on precleaned glass microscope slides (Fisherbrand Superfrost/Plus; Fisher Scientific), fixed with 100% methanol for 20 minutes at room temperature, and air dried at room temperature. 30 Photographs of tissue sections were taken with a digital camera mounted on an inverted microscope (Nikon, Tokyo, Japan). The areas of tissue sections were calculated with NIH Image software (Scion Image 1.60; Scion, Frederick, MD), and volumes were calculated by multiplying tissue section areas by section thickness. Enzymatic Digestion of KS and CS/DS Tissue sections (n ϭ 5), each from a cornea of a separate chick embryo incubated to the same age, were placed on glass slides, encircled with a hydrophobic ring (PAP pen), and then covered with a droplet of digestion solution, as follows: for analysis of KS sulfated disaccharides, the digestion solution consisted of 25 L of 0.1 M ammonium acetate buffer (pH 6.0), 0.1 mU/L keratanase II, and 50 pmol/L ⌬di-2S (as an internal standard) applied to each section. For analysis of CS/DS sulfated disaccharides, the digestion solution consisted of 25 L containing 50 mM ammonium acetate buffer (pH 8.0), chondroitinase ABC (1 mU/L), and ⌬UA-␤-1,4-GlcNS (as an internal standard, 50 pmol/L) applied on each section. The slides were then incubated in a moist chamber at 37°C for 24 hours. The solution was carefully collected from each tissue section, and its enzyme activity was terminated by heating for 10 minutes at 100°C. Digest solution (10 L) was diluted by adding 70 L MeOH, 5 L of 5 mM ammonium acetate buffer (pH 7.5), 5 L of 2 mM (NH 4 ) 2 SO 4 , and 10 L of water, Mass Spectrometry Mass spectra were obtained using an electrospray ionization source on a quadrupole ion trap instrument (Esquire 3000; Bruker Daltonics, Billerica, MA). Mass spectra were obtained in negative ion mode. The spray voltage was 3.5 kV. Dry gas (nitrogen) flowed at 5.0 L/min, and the drying temperature was 180°C. The mass range scanned was m/z 50 to 600. These same instrument conditions were used for all standards, mock mixtures, and digestion samples. Data acquisition software was used to record the results (Data Analysis 3.0; Bruker Daltonics). Quantitative Analysis of KS Sulfated Disaccharides A single-point normalization factor method, as described elsewhere, Quantitative Analysis of CS/DS Sulfated Disaccharides The total molar concentration of ⌬di-4S plus ⌬di-6S was determined by quantitative analysis of the intensity of the characteristic molecular ion at m/z 458.1, using an internal standard, ⌬UA-␤-1,4-GlcNS and a singlepoint normalization factor, similar to the quantitative analysis of KS sulfated disaccharides just described. The R value determined for the total concentration of ⌬di-4S and ⌬di-6S was 1.2 Ϯ 0.3. The relative molar percentages of ⌬di-4S and ⌬di-6S isomers in a mixture were calculated from the observed relative intensities of the diagnostic ions for each isomer in the MS 2 spectrum of that mixture, using a system of two equations that correct for the presence of additional ions in the MS 2 spectrum, as described elsewhere. where A and B are the apparent percentages of ⌬di-4S and ⌬di-6S in any unknown mixture, and C 299.9 and C 281.8 are the percentage contributions of the two diagnostic ions in the mixture. However, because many factors influence peak intensities in a mixture, including differences in the respective ionization efficiencies of the isomers, 22 further normalization factors, R 4S and R 6S , must be used. To determine these normalization factors, the percentage contribution of each diagnostic ion was determined from the MS 2 spectrum of a 1:1 mixture of ⌬di-4S and ⌬di-6S, as described in RESULTS Analysis of KS Standard Disaccharides The mass spectra of KS MSD and DSD are shown in MSD yielded one singly charged ion [M Ϫ H] Ϫ at m/z 462.0 Ϫ at m/z 183.8, and [OCHCH 2 OSO 3 ] Ϫ at m/z 138.8 ( Ϫ , and at m/z 299.9, corresponding to Y 1 , representing the occurrence of glycosidic cleavage, with no ring cleavage found in this case Analysis of CS/DS Sulfated Disaccharides The three CS/DS sulfated disaccharide isomers, ⌬di-2S, ⌬di-4S, and ⌬di-6S, yielded very simple MS 1 spectra. The most abundant ion in each case (Figs. 3A-C) corresponds to molecular ion [M Ϫ H] Ϫ for each disaccharide with one sulfate, all of which generate the same molecular ion [M Ϫ H] Ϫ at m/z 458.1. (Y 1 ions) for ⌬di-4S Detection of Sulfated Disaccharides from KS of Embryonic Chick Corneas Detection of Sulfated Disaccharides from CS/DS of Embryonic Chick Corneas Content of the KS Sulfated Disaccharides MSD and DS

v8a48-tasheva pgmkr

Purpose: To study the role of mimecan, a member of the small leucine-rich proteoglycans (SLRPs) gene family and one of the major components of the cornea and other connective tissues, mice that lack a functional mimecan gene were generated and characterized. Methods: Mimecan-deficient mice were generated by gene-targeting using standard techniques. Mice were genotyped by Southern blot analysis. The absence of mimecan transcripts was confirmed by Northern blot analysis. Corneal clarity was examined by slit lamp biomicroscopy. The strength of the skin was evaluated using a biomechanical skin fragility test. Collagen morphology in cornea and skin preparations from mimecan-null and control wild-type mice was analyzed by transmission electron microscopy. The diameter of collagen fibrils in these tissues was determined by morphometric analysis. Results: Mice lacking mimecan appear to develop normally, are viable and fertile. In a controlled laboratory environment they do not display an evident pathological phenotype compared to wild type mice. Examination of corneal clarity and measurements of corneal thickness show no significant changes in the cornea. However, a skin fragility test revealed a moderate reduction in the tensile strength of skin from mutant mice. Ultrastructural analyses show, on average, thicker collagen fibrils in both corneal and skin preparations from mimecan-null mice. Collagen fibrils from the cornea of mutant mice show an average diameter of 31.84±0.322 nm, versus 22.40±0.296 nm in their wild type litter-mates. The most pronounced increase in collagen fibril diameter was found in the skin of mimecan-null mice, who demonstrated an average diameter of 130.33±1.769 nm, versus 78.82±1.157 nm in the wild type mice. In addition, size variability and altered collagen morphology was detected in dorsal and tail skin preparations from the mutant mice. Conclusions: The results of the present study demonstrate that mimecan, similar to other members of the SLRP gene family, has a role in regulating collagen fibrillogenesis in vivo. Further studies, such as functional challenges, an evaluation of potential compensation by other proteins (including members of the SLRP family), and generation of doubleknockouts will be necessary to fully uncover physiological functions of mimecan in mice

Expression studies of osteoglycin/mimecan (OGN) in the cochlea and auditory phenotype of Ogn-deficient mice

Genes involved in the hearing process have been identified through both positional cloning efforts following genetic linkage studies of families with heritable deafness and by candidate gene approaches based on known functional properties or inner ear expression. The latter method of gene discovery may employ a tissue- or organ-specific approach. Through characterization of a human fetal cochlear cDNA library, we have identified transcripts that are preferentially and/or highly expressed in the cochlea. High expression in the cochlea may be suggestive of a fundamental role for a transcript in the auditory system. Herein we report the identification and characterization of a transcript from the cochlear cDNA library with abundant cochlear expression and unknown function that was subsequently determined to represent osteoglycin (OGN). Ogn-deficient mice, when analyzed by auditory brainstem response and distortion product otoacoustic emissions, have normal hearing thresholds