Elena S. Tasheva

Mimecan, the 25-kDa Corneal Keratan Sulfate Proteoglycan, Is a Product of the Gene Producing Osteoglycin

Bovine cornea contains three unique keratan sulfate proteoglycans (KSPGs), of which two (lumican and keratocan) have been characterized using molecular cloning. The gene for the third protein (KSPG25) has not been identified. This study examined the relationship between the KSPG25 protein and the gene for osteoglycin, a 12-kDa bone glycoprotein. The N-terminal amino acid sequence of KSPG25 occurs in osteoglycin cDNA cloned from bovine cornea. The osteoglycin amino acid sequence makes up the C-terminal 47% of the deduced sequence of the KSPG25 protein. Antibodies to osteoglycin reacted with intact corneal KSPG, with KSPG25 protein, and with a 36-kDa protein, distinct from lumican and keratocan. KSPG25-related proteins, not modified with keratan sulfate, were also detected in several connective tissues. Northern blot analysis showed mRNA transcripts of 2.4, 2.5, and 2.6 kilobases in numerous tissues with the 2.4-kilobase transcript enriched in ocular tissues. Ribonuclease protection analysis detected several protected KSPG25 mRNA fragments, suggesting alternate splicing of KSPG25 transcripts. We conclude that the full-length translation product of the gene producing osteoglycin is a corneal keratan sulfate proteoglycan, also present in many non-corneal tissues without keratan sulfate chains. The multiple size protein products of this gene appear to result from in situ proteolytic processing and/or alternative splicing of mRNA. The name mimecan is proposed for this gene and its products.

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 locus 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 stenosis.

Differential splicing and alternative polyadenylation generate multiple mimecan mRNA transcripts.

We previously showed the 25-kDa corneal keratan sulfate proteoglycan to be a translation product of the gene producing osteoglycin and proposed the name mimecan for this gene and its product. We also demonstrated three mimecan RNA transcripts using Northern blot analysis. In this report, we investigate the mechanisms accounting for these transcripts. Ribonuclease protection analysis and reverse transcription-polymerase chain reaction of bovine corneal mRNA detected a mimecan transcript that lacked 278 base pairs of the 5′-untranslated region between residues 62 and 340. This splice variant represents the predominant form of mimecan mRNA in bovine cornea and sclera. It was also detectable in other bovine tissues as a minor transcript. Two additional cDNA clones that were isolated contained 398 bases of nucleotide sequence at the 3′-end of mimecan cDNA, not present in the published sequence. Ribonuclease protection analyses with the 3′-probe, which included the new sequence, allow detection of three RNA transcripts while 5′-probes recognized only two. These results indicate that the three canonical polyadenylation sites in the 3′-untranslated region of mimican mRNA are alternatively selected. Possible roles for this previously undetected degree of diversity of mimecan RNA isoforms transcribed in the same tissue are discussed.

Transcriptional activation of bovine mimecan by p53 through an intronic DNA-binding site

Mimecan is a small leucine-rich proteoglycan that can occur as either keratan sulfate proteoglycan in the cornea or as glycoprotein in many connective tissues. As yet, there is no information on its transcriptional regulation. Recently we demonstrated the presence of eight mimecan mRNA transcripts generated by alternative transcription initiation, alternative polyadenylation, and differential splicing, all of which encode an identical protein. Here we report a conserved consensus p53-binding DNA sequence in the first intron of bovine and human mimecan genes and show that wild-type p53 binds to this sequence in vitro. Co-transfections of Saos-2, HeLa, NIH 3T3, and primary bovine corneal keratocytes with bovine mimecan promoter/luciferase reporter constructs in combination with p53 expression vectors activate the second mimecan promoter through the p53-binding sequence. In addition, we show absence of mimecan expression in different tumors and cancer cell lines, where p53 frequently is inactivated/mutated. Thus, this work provides novel information that links mimecan to the p53 network.

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

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 the ECM. This study asked which of the proteins that may regulate keratocytes or corneal nerve growth cone immigration interact with corneal GAGs. METHODS Biotinylated KS (bKS), CSA (bCSA), and HA (bHA) were prepared and used in microarray protocols to assess their interactions with 8268 proteins and a custom microarray 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 were determined. RESULTS Highly sulfated KS interacted with 217 microarray proteins, including 75 kinases, several membrane or secreted proteins, many cytoskeletal proteins, and many nerve function proteins. CSA interacted with 24 proteins, including 10 kinases and 2 cell surface proteins. HA interacted with 6 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 nine proteins, including ROBO2, 2 EPHs, 1 EFN, two SEMAs, and netrin 4. HA bound no ECM nerve-related epitopes. SPR confirmed that KS binds SLIT2 strongly. The KS core protein mimecan/osteoglycin bound 15 proteins. CONCLUSIONS Corneal stromal GAGs bind, and thus could alter the availability or conformation of, many proteins that may influence keratocyte and nerve growth cone behavior in the cornea.

Analysis of the promoter region of human mimecan gene.

Mimecan is a small leucine-rich proteoglycan (SLRP) that may play an important role in the regulation of cellular growth as illustrated by ability of growth factors and cytokines to modulate its expression and by recent demonstration that bovine mimecan is transcriptionally activated by p53 through a conserved intronic recognition site. To investigate transcriptional regulation of human mimecan, the upstream region and the first intron of this gene were cloned and analyzed. Within a 296-bp upstream region required for basal gene expression, there are three initiator (Inr) elements, an E-box and Oct-1, metal response element (MRE), and NF-kappa B recognition sites. Upstream stimulatory factor (USF)-1, Oct-1 and MRE-binding proteins were identified as proteins that bind to these regulatory elements and support transcription of mimecan in MG-63 cells. The first intron of human mimecan contains enhancer and silencer elements. Reporter gene transfections demonstrated that cooperation of upstream region and intronic enhancer elements are required for maximal gene expression in both p53-deficient and wild-type p53-expressing cells. Within the footprinted intronic enhancer region an interferon-stimulated response element (ISRE) is present. Using electrophoretic mobility shift assay (EMSA), interferon regulatory factor (IRF)-1 was identified as a protein that binds to this region in MG-63 but not in U-937 cells. In vitro translated IRF-1 also was shown to bind to this ISRE. These results demonstrate that the first intron of human mimecan gene carries important regulatory elements, including p53 DNA-binding site and ISRE, and should promote a better understanding of molecular bases for cell type-specific regulation of mimecan transcription.

Structure and Sequence of the Gene Encoding Human Keratocan

Keratocan is one of the three major keratan sulfate proteoglycans characteristically expressed in cornea. We have isolated cDNA and genomic clones and determined the sequence of the entire human keratocan (Kera) gene. The gene is spread over 7.65 kb of DNA and contains three exons. An open reading frame starting at the beginning of the second exon encodes a protein of 352 aa. The amino acid sequence of keratocan shows high identity among mammalian species. This evolutionary conservation between the keratocan proteins as well as the restricted expression of Kera gene in cornea suggests that this molecule might be important in developing and maintaining corneal transparency.

The Bovine Mimecan Gene MOLECULAR CLONING AND CHARACTERIZATION OF TWO MAJOR RNA TRANSCRIPTS GENERATED BY ALTERNATIVE USE OF TWO SPLICE ACCEPTOR SITES IN THE THIRD EXON

Mimecan is a proteoglycan expressed by many connective tissues. It was originally isolated in a truncated form as a bone-associated glycoprotein, osteoglycin, and was considered an osteoinductive factor. Recently, we demonstrated that the full-length translation product of the cDNA encoding mimecan is a corneal keratan sulfate proteoglycan present in other tissues without keratan sulfate chains. We also described multiple mimecan mRNA transcripts generated by differential splicing and alternative polyadenylation. In this study, we isolated genomic clones and determined the genomic organization of the bovine mimecan gene. The gene is spread over >33 kilobases of continuous DNA sequence and contains eight exons. The newly discovered first exon, identified by 5′-rapid amplification of cDNA ends, consists of a 5′-untranslated region and is enriched in C+G nucleotides. Two transcription initiation sites starting at the first and at the second exons were determined by primer extension. Molecular characterization shows that alternatively spliced RNA isoforms are generated by the use of two distinct splice acceptor sites in the third exon situated 278 base pairs apart. We determined a partial genomic structure of the human mimecan gene and demonstrated two alternatively spliced RNA transcripts that are generated likewise. Despite the diversity of mimecan transcripts, the primary structure of the core protein is encoded from exons 3 to 8 and remains unchanged, indicating its functional importance. Using ribonuclease protection assay, we analyzed the patterns of spliced RNA expressed in cultured bovine keratocytes. We demonstrated that their expression is differentially modulated in a temporal manner by basic fibroblast growth factor.

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.

A densely methylated DNA Island is associated with a chromosomal replication origin in the humanRPS14 locus

We describe a 258-bp densely methylated DNA island (DMI) and chromosomal origin of bidirectional DNA replication within the transcribed portion of the human RPS14 intron 1. Together with the DMIs previously detected in two functional Chinese hamster replication origins [see Ref. 1, pp. 5636–5644], observations described in this report strengthen the correlation between densely methylated DNA islands and active mammalian chromosomal replication origins. Accordingly, DMIs may prove to be reliable physical markers for origins of bidirectional DNA replication in complex genomic DNAs of higher animals.