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K Doege

Publications and source records attributed to K Doege.

17 recordsLinked to original sources
PubMed2001

Calreticulin, PDI, Grp94 and BiP chaperone proteins are associated with retained COMP in pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP), a large pentameric glycoprotein and member of the thrombospondin (TSP) group of extracellular proteins, is found in the territorial matrix surrounding chondrocytes. More than 50 unique COMP mutations have been identified as causing two skeletal dysplasias: pseudoachondroplasia (PSACH); and multiple epiphyseal dysplasia (EDM1). Recent studies suggest that calcium-binding and calcium-induced protein folding differ between wild type and mutant proteins, and abnormal processing of the mutant COMP protein contributes to the characteristic enlarged lamellar appearing rER cisternae in PSACH and EDMI chondrocytes in vivo and in vitro. Towards the goal of delineating the pathogenesis of PSACH and EDM1, in-vivo PSACH growth plate and in-vitro PSACH chondrocytes cultured in alginate beads were examined to identify and localize the chaperone proteins participating in the processing of the retained extracellular matrix proteins in the PSACH rER. Aggrecan was localized to both the rER cisternae and matrix while COMP and type IX collagen were only found in the rER. Type II collagen was solely found in the ECM suggesting that it is processed and transported differently from other retained ECM proteins. Five chaperone proteins: BiP (Grp78); calreticulin (CRT); protein disulfide (PDI); ERp72; and Grp94, demonstrated immunoreactivity in the enlarged PSACH cisternae and the short rER channels of chondrocytes from both in-vivo and in-vitro samples. The chaperone proteins cluster around the electron dense material within the enlarged rER cisternae. CRT, PDI and GRP94 AB-gold particles appear to be closely associated with COMP. Immunoprecipitation and Western blot, and Fluorescence Resonance Energy Transfer (FRET) analyses indicate that CRT, PDI and GRP94 are in close proximity to normal and mutant COMP and BiP to mutant COMP. These results suggest that these proteins play a role in the processing and transport of wild type COMP in normal chondrocytes and in the retention of mutant COMP in PSACH chondrocytes.

Achondroplasia↗
PubMed2000

The intermediates of aggrecanase-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1.

We have examined the abundance and structure of intermediates in the chondrocyte-mediated degradation of aggrecan by aggrecanase(s). Degradation products were identified by Western-blot analysis with antibodies to cleavage-site neoepitopes and to peptides within the globular domains. Rat chondrosarcoma tumour contained full-length aggrecan and all of the individual peptides expected from single independent cleavages at each of the four aggrecanase sites in the chondroitin sulphate (CS) domain. Kinetic analysis of the products present in rat chondrosarcoma cell cultures treated with interleukin-1b showed that the first aggrecanase-mediated cleavages occurred at the four sites within the CS attachment region to generate two stable intermediates, Val(1)-Glu(1459) and Val(1)-Glu(1274). These species were subsequently cleaved at the Glu(373) site in the interglobular domain to form the terminal products, Val(1)-Glu(373), Ala(374)-Glu(1274) and Ala(374)-Glu(1459). It therefore appears that the aggrecanase-mediated processing of native aggrecan by chondrocytes in situ is initiated within the CS-attachment region and completed by cleavage within the interglobular domain. Since it has been shown that digestion of aggrecan monomer in solution with recombinant ADAMTS-4 [Tortorella, Pratta, Liu, Austin, Ross, Abbaszade, Burn and Arner (2000) Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J. Biol. Chem. 275, 18566-18573] exhibits similar kinetics, it appears that preferential proteinase cleavage in the CS-rich region is determined by properties inherent in the aggrecan monomer itself, such as preferred peptide sequences for enzyme binding or enhanced accessibility to the core protein at these sites.

Aggrecans↗
PubMed1998

An association between an aggrecan polymorphic allele and bilateral hand osteoarthritis in elderly white men: data from the Baltimore Longitudinal Study of Aging (BLSA).

OBJECTIVE: The aggrecan proteoglycan is a major component of articular cartilage and supports the biomechanical function of this tissue. A variable number tandem repeat (VNTR) polymorphism has been discovered recently in a region of the human aggrecan gene that codes for the chondroitin sulfate attachment sites. We examined whether alleles of this polymorphism displayed a non-random association with bilateral hand or knee osteoarthritis (OA) in men from the Baltimore Longitudinal Study of Aging (BLSA). DESIGN: DNA was obtained from 93 Caucasian men, aged 60 and above, who had bilateral hand and standing knee radiographs read for changes of OA. The DNA was analyzed by polymerase chain reaction (PCR) and/or Southern blotting for the presence of the VNTR alleles. RESULTS: Bilateral hand OA and knee OA were present in 46 and 30% of the men respectively. The following distribution of alleles was observed: allele 33 (0.5%), 29 (2.2%), 28 (31.7%), 27 (43.0%), 26 (16.7%), 25 (3.2%), 22 (2.2%) and 19 (0.5%). This distribution was similar to that detected in a random population of individuals from a separate study. In multiple logistic regression analysis, adjusting for age and body mass index, the presence of allele 27 was associated with bilateral hand OA with an odds ratio (OR) = 3.23 (95% confidence intervals (CI): 1.24-8.41). No other alleles showed an association with bilateral hand OA and the association between allele 27 and bilateral knee OA was not statistically significant (OR = 1.14; 95% CI: 0.45-2.88). CONCLUSIONS: These data demonstrate the first association between a human aggrecan gene polymorphic allele and hand OA. This finding supports the concept that genetic factors may play a role in the development and/or progression of some forms of age-onset OA.

Aged↗
PubMed1997

Non-glycosaminoglycan bearing domains of perlecan and aggrecan influence the utilization of sites for heparan and chondroitin sulfate synthesis.

Perlecan and aggrecan are proteoglycans that receive primarily heparan sulfate and chondroitin sulfate side chains, respectively. Their large multidomained core proteins have little or no homology to each other and their glycosaminoglycan (GAG) attachment sites are restricted to certain domains only. We examined the involvement of the non-GAG bearing domains in designating the GAG type added to the GAG attachment domain by preparing cDNA constructs that expressed perlecan/aggrecan chimeras as recombinant products in COS-7 cells and then determining the size and GAG composition of the recombinant products. The results showed that domain I of perlecan receives primarily (73-81%) heparan sulfate when coupled with domain II and III of perlecan, but when coupled with the G3 domain of aggrecan, it receives primarily (59-63%) chondroitin sulfate. Furthermore, the chondroitin sulfate attachment region of aggrecan received GAG side chains more readily when coupled to the G3 domain of aggrecan than when coupled to domains II and III of perlecan. The GAG side chains on all these recombinant products were small and similar in size. These findings indicate that the utilization of attachment sites for heparan and chondroitin sulfate or the sulfation of these GAGs can be influenced, in part, by non-GAG bearing domains.

Aggrecans↗
PubMed1995

Mouse aggrecan, a large cartilage proteoglycan: protein sequence, gene structure and promoter sequence.

Seven genomic clones for mouse aggrecan core protein have been isolated including 3 kb of 5'- and 7 kb of 3'-flanking sequences. All exon sequences and their intron boundary sequences in these clones were identified and mapped by DNA sequencing. The gene spans at least 61 kb and contains 18 exons. Exon 1 encodes 5'-untranslated sequence and exon 2 contains a translation start codon, methionine. The coding sequence is 6545 bp for a 2132-amino-acid protein with calculated M(r) = 259,131 including an 18-amino-acid signal peptide. There is a strong correlation between structural domains and exons. Notably, the chondroitin sulphate domain consisting of 1161 amino acids is encoded by a single exon of 3.6 kb. Although link protein has similar structural domains and subdomains, the sequence identity and the organization of exons encoding the subdomains B and B' of G1 and G2 domains revealed a strong similarity of mouse aggrecan to both human versican and rat neurocan. Primer extension analysis identified four transcription start sites which are close together. The promoter sequence showed high G/C content (65%) and contained several consensus binding motifs for transcription factors including Sp-1 and the glucocorticoid receptor. There are stretches of sequences similar to the promoter region of both the type-II collagen and link protein genes. These sequences may be important for cartilage gene expression.

Aggrecans↗
PubMed1993

Alternate exon usage is a commonly used mechanism for increasing coding diversity within genes coding for extracellular matrix proteins.

Extracellular matrix proteins are a diverse family of secreted proteins and glycoproteins that are responsible for a variety of critical functions in different tissues. A large number of multiexon genes encode these proteins of the extracellular matrix. Over the last few years, it has become evident that the processing of the pre-mRNA from several of these genes involves alternative splicing. This review summarizes the known examples of alternative splicing in genes coding for the extracellular matrix and attempts to relate the increase in coding diversity generated by alternate exon usage to the function(s) of individual extracellular matrix proteins.

Alternative Splicing↗
PubMed1991

Characterization of the promoter for the rat and human link protein gene.

We have isolated the 5'-end of the gene for the rat and human link protein by screening genomic libraries with oligonucleotides corresponding to the 5'-cDNA sequence. Several overlapping clones were isolated for the human link protein gene, while only one clone was obtained for the rat. All the clones contained a single exon of which the sequence was identical to the most 5'-end of the rat and human cDNAs. Transcription initiation sites for the rat link gene were identified by primer extension and S1 protection analysis using total RNA from the rat Swarm chondrosarcoma. Transcriptional initiation sites for the human link gene were determined by specific primer extension of RNA from human fetal cartilage. Comparison of 1500 bp of 5'-flanking sequence between the rat and human link protein genes showed strong sequence conservation near the start site of transcription with 80% overall identity. Analysis of the 5'-flanking regions also revealed a large inverted repeat consisting of repeating purine-pyrimidine, which has the potential to form left-handed Z-DNA. Transcriptional regulation of the link protein gene was studied by coupling either 7.0 kb or 0.85 kb of 5'-flanking rat DNA to the chloramphenicol acetyltransferase (CAT) gene followed by transfection into chick embryonic chondrocytes (CEC) and HeLa cells. Both constructs had considerable CAT activity in CEC cells and less activity in HeLa cells. Furthermore, inclusion of a DNA fragment from the first intron increased relative CAT activity in both of these cell types. The increased activity from the first intron was shown to be orientation independent in CEC. These results indicate the presence of positive cisacting regulatory elements in both the promoter and first intron of the rat gene for link protein.

Animals↗
PubMed1991

Analysis of the chondroitin sulfate proteoglycan core protein (CSPGCP) gene in achondroplasia and pseudoachondroplasia.

Achondroplasia and pseudoachondroplasia are autosomal dominant skeletal dysplasias resulting in short-limbed dwarfism. Histologic and ultrastructural studies of the cartilage in pseudoachondroplasia and in homozygous achondroplasia have suggested a structural abnormality in chondroitin sulfate proteoglycan (CSPG), a major structural protein in the extra-cellular matrix. The gene encoding CSPG core protein (CSPGCP) is thus a logical "candidate gene" for analysis in these conditions. cDNA probes encoding CSPGCP were used to identify restriction fragment length polymorphisms (RFLPs) in DNA from a panel of control individuals. No gross alterations at the CSPGCP locus were noted in DNA from 37 individuals with achondroplasia and 5 individuals with pseudoachondroplasia. In addition, allelic frequencies of the RFLPs were not significantly different among controls and patients with either condition. In one three-generation family with achondroplasia, close linkage of the CSPGCP locus and the skeletal dysplasia was excluded using a Bgl II polymorphism. Similarly, in a three-generation family with pseudoachondroplasia, the CSPGCP gene was not tightly linked to the disease phenotype. These results indicate that mutations at the chondroitin sulfate proteoglycan core protein locus do not cause achondroplasia or pseudoachondroplasia in these families.

Achondroplasia↗
PubMed1988

A segment of the cartilage proteoglycan core protein has lectin-like activity.

A segment of 130 residues near the COOH terminus of the proteoglycan core protein derived from rat cartilage is highly homologous to the carbohydrate-recognition domain of the chicken hepatic lectin and other vertebrate carbohydrate-binding proteins. This portion of the protein has been expressed in an in vitro transcription and translation system and has been tested for its ability to interact with carbohydrates using affinity chromatography on immobilized sugars. A distinct specificity of the binding interaction is demonstrable, with fucose and galactose being the preferred ligands. However, the affinity of the expressed domain of the proteoglycan core protein is lower than that of the other known binding domains, since it elutes from the columns even in the presence of Ca2+.

Aggrecans↗
PubMed1988

Alternative splicing generates two different mRNA species for rat link protein.

Previously we isolated a cDNA clone which encoded the carboxyl-terminal portion of the rat link protein (Doege, K., Hassell, J.R., Caterson, B., and Yamada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3761-3765). Here we report the isolation of cDNA clones encoding the remainder of the protein. Sequence analysis revealed two classes of cDNA clones identical except for the occurrence of an additional internal nucleotide segment of 159 base pairs coding for 53 amino acids in one of the clones. Genomic clones were then isolated using the cDNA as a probe. Sequence analysis of genomic clones revealed that the internal 159-base pair cDNA segment was contained in a single exon. The protein sequence deduced from these two cDNAs were Mr = 44,779 (link 45) and Mr = 38,570 (link 39). Northern blotting of chondrosarcoma RNA revealed that both the alternate exon specific to link 45 and an exon common to both link 45 and 39 hybridized to the same size classes of link protein transcripts (1.5-5.5 kilobases), although link 45 transcripts are much less prevalent than link 39 transcripts. The link 39 sequence corresponds exactly with the predominant form of chondrosarcoma link protein. The similarity in size between link 45 and the larger form of link protein (LP1) observed in other species appears fortuitous.

Amino Acid Sequence↗
PubMed1988

An established rat cell line expressing chondrocyte properties.

Chondrocytes express a well-characterized set of marker proteins making these cells useful for studies on differentiation and regulation of gene expression. Because of the inherent instability of primary rat chondrocytes in culture, and because several rat chondrocyte genes have been cloned and characterized (including the collagen II promoter and enhancer), a rat chondrocyte cell line would be especially useful. To obtain this line we infected primary fetal rat costal chondrocytes with a recombinant retrovirus (NIH/J-2) carrying the myc and raf oncogenes, which have been shown to have an "immortalizing" function. Following infection, a rapidly proliferating clonal line was isolated that maintained a stable phenotype through 45 passages (11/2 year in culture). This line, termed IRC, grows in suspension culture as multicellular aggregates and in monolayer culture as polygonal cells which accumulate an alcian blue-stainable matrix. IRC cells synthesize high levels of cartilage proteoglycan core protein, and link protein, but show reduced collagen II expression. In addition, the cells express virally derived myc mRNA and protein, but do not express v-raf. Retinoic acid, which is a known modulator of chondrocyte phenotype, down-regulates expression of chondrocyte marker proteins, while stimulating v-myc expression by IRC cells. These data suggest that v-myc expression by chondrocytes results in rapid cell division and maintenance of many aspects of the differentiated phenotype. These "immortalized" cells, however, remain responsive to agents such as retinoic acid which modulate cell phenotype. The potential exists for development of chondrocyte cell lines from diseased cartilage, as well as from human cartilage.

Animals↗
PubMed1987

Complete primary structure of the rat cartilage proteoglycan core protein deduced from cDNA clones.

We have obtained overlapping cDNA clones for the entire coding sequence of the rat cartilage proteoglycan core protein from the Swarm rat chondrosarcoma. These cDNAs hybridize to two sizes of RNA transcripts of 8.2 and 8.9 kilobase pairs, which contain large 3'-untranslated sequences. The total contiguous cDNA is 6.55 kilobase pairs in size, and codes for a 2124-residue protein, including a 19-residue signal peptide. The sequence forms a series of eight structural domains including two globules, (Mr = 37,000 and 22,000) at the NH2 terminus of the molecule, one a complete and one a partial copy of the cartilage link protein. The major feature of the deduced protein sequence is a 1,104-residue segment containing 117 Ser-Gly sequences, the presumed chondroitin sulfate attachment sites. These are arranged in three domains of 428, 503, and 173 amino acids. The first domain contains 11 complete or partial repeats of a 40-residue unit, and the second domain is composed of six copies of a 100-residue repeating sequence. The first pattern is the more highly conserved, and may have given rise to the second. The carboxyl-terminal domain is a third globule which has homology with animal lectins.

Aggrecans↗
PubMed1986

Partial cDNA sequence encoding a globular domain at the C terminus of the rat cartilage proteoglycan.

We have isolated and sequenced a cDNA clone of 872 base pairs from the 3' end of the mRNA for the large cartilage specific proteoglycan from rat. Identification was confirmed by a comparison with published protein sequence. Hybridization analysis shows the presence of an 8-9-kilobase mRNA for this proteoglycan in rat and chick sternal chondrocytes and rat chondrosarcoma cells, but not in RNA from rat fibroblasts, vitamin A-treated chick chondrocytes, chick crop, or bone. The carboxyl portion of the proteoglycan is deduced to terminate in a globular domain, which includes a region homologous to a chick hepatic lectin, and is possibly involved in binding to N-acetylglucosamine. The clone extends into a region where serines are clustered, probably the start of the chondroitin sulfate-rich region.

Amino Acid Sequence↗
PubMed1986

Link protein cDNA sequence reveals a tandemly repeated protein structure.

Link protein stabilizes the cartilage proteoglycan/hyaluronic acid aggregate by binding to both components. We screened a cDNA library prepared from rat chondrosarcoma mRNA in the lambda gt11 expression vector with monoclonal antibodies and polyclonal antisera to link protein. We obtained a clone for two-thirds of the link protein cDNA and identified it based on its deduced amino acid sequence. There are four RNA transcripts for link protein, ranging from 1.5 to 5.5 kilobases in size. The deduced amino acid sequence for link protein shows two domains of 100 residues each, which share 44% homology; within each domain is a 19-residue stretch 74% homologous with its counterpart. This structure indicates that link protein may have been formed by gene duplication.

Amino Acid Sequence↗
PubMedDate not supplied

Expression and structure of cartilage proteins.

Cartilage has unique physical characteristics attributable to the presence of an unusually high content of proteoglycan embedded in the network of collagen fibrils. Advances in understanding the structure of these components and how their synthesis is regulated have been greatly assisted by the application of molecular biology. For example, an immortalized rat chondrocyte cell line was obtained by infection with a recombinant retrovirus encoding the myc gene product. Several positive and negative DNA regulatory elements of the collagen II gene have been identified that appear to be important in the regulation of this gene in chondrocytes. The complete primary structure of the cartilage proteoglycan (aggrecan) core protein deduced from cDNA sequence displays a complex multidomain structure including numerous repeats of Ser-Gly sequences and sequence homologies with link protein and animal lectins. Such studies advance our understanding of normal morphogenetic events and lay the groundwork for determining the basis of molecular and genetic defects.

Animals↗