Biomedical subjects

T D Huynh

Publications and source records attributed to T D Huynh.

5 recordsLinked to original sources

PubMed1997

Complete coding sequence of bovine aggrecan: comparative structural analysis.

The previously available sequence for bovine aggrecan included only the KS domain, the C-terminal portion of the CS-2 domain, and the entire CS-3 and G3 domains. We have isolated cDNA clones for previously uncharacterized portions of the bovine aggrecan sequence, and, when we combined them with previously published incomplete sequences, have obtained a complete sequence for the entire core protein. The bovine aggrecan sequence, which is a composite of new sequence data and previously published incomplete sequences, is 2327 residues in length. Although there is significant conservation of G1, G2, and G3 globular domains between species, there are differences in the length of the interglobular domain, in the number of KS domain hexapeptide repeats and CS domain repeats, and in alternative splicing within the G3 domain. The bovine aggrecan KS domain contains 24 repeats of a hexapeptide motif. The largely uncharacterized CS-1 domain of bovine aggrecan was found to contain 27 variable repeats of a 21-residue consensus sequence. A notable feature of the bovine CS-1 domain is in the distribution of single Ser-Gly dipeptides, the majority of which are separated by 7 or 8 amino acids, compared to the human, where discrete pairs of Ser-Gly dipeptides are separated by 13 amino acids. The CS-2 domain contains a total of six "homology domains" with 4 complete and 2 partial approximately 100-residue repeats. Each "homology domain" contains a "nodal" region with few sites for CS chain addition that is highly conserved between species, suggesting a possible role in aggrecan biosynthesis or catabolism.

Aggrecans↗

PubMed1996

Purification and characterization of decorin core protein expressed in Escherichia coli as a maltose-binding protein fusion.

Decorin is a small leucine-rich proteoglycan which is a component of the extracellular matrix of many connective tissues. We have developed a method for expression and purification of decorin as a fusion protein in Escherichia coli. A PCR product coding for the 330-amino-acid-residue secreted form of bovine decorin core protein was cloned into the vector pMal-c for expression in E. coli as a maltose-binding protein (MBP) fusion. Expressed MBP-decorin tended to form insoluble aggregates resistant to degradation by E. coli intracellular proteases. Fusion protein was therefore solubilized from bacterial inclusion bodies using guanidine HCl and refolded by dilution of the chaotrope to minimally denaturing conditions and disulfide shuffling. Final purification included amylose resin affinity chromatography and size exclusion chromatography. The 79-kDa MBP-decorin fusion protein could be completely cleaved by factor Xa protease in 24 h to yield 43-kDa MBP and 36-kDa decorin core protein. The decorin core protein was separated from MBP and factor Xa protease by DEAE-Sephacel chromatography. Using a solid-phase assay, we have characterized its binding affinity for extracellular matrix components known to interact with tissue-derived decorin including collagen type VI, collagen type I, and fibronectin. The purification and refolding protocol described here may be generally applicable to bacterial expression of other members of this growing family of related small leucine-rich proteoglycan core proteins.

ATP-Binding Cassette Transporters↗

PubMed1995

Expression and characterization of a single recombinant proteoglycan tandem repeat domain of link protein that binds zinc and hyaluronate.

The first proteoglycan tandem repeat (PTR) of bovine link protein has been cloned in the pMAL-c vector and overexpressed in fusion with maltose-binding protein (MBP) in Escherichia coli. The fusion protein can be isolated from the soluble phase of the bacterial lysate by amylose affinity chromatography. The PTR domain can be cleaved from the MBP domain with factor Xa protease. Evidence using zinc affinity chromatography is presented which indicates that at least one of the zinc-binding sites of bovine link protein is contained within the first PTR domain. Zinc affinity chromatography was then incorporated as the final purification step of the MBP/PTR protein. Evidence for the binding of MBP/PTR to hyaluronic acid (HA) is demonstrated by coprecipitation with HA using cetylpyridinium chloride. Binding is specific since MBP/PTR does not coprecipitate with chondroitin sulfate. Binding is also demonstrated in an ELISA assay on HA-coated plates. In this assay, binding could be inhibited by the addition of HA or HA oligosaccharides.

Amino Acid Sequence↗

PubMed1995

Bovine chondrocyte link protein cDNA sequence: interspecies conservation of primary structure and mRNA untranslated regions.

The sequence for bovine link protein cDNA, including 108 bases of the 5' untranslated region (UTR) and 768 nucleotides of the 3' UTR, was determined from polymerase chain reaction products and bovine articular chondrocyte cDNA clones. The deduced primary structure for bovine link protein predicts a protein 354 amino acid residues in length. Comparative analysis with link protein sequence from several other species revealed overall high conservation of protein coding sequence. High nucleotide sequence conservation was observed within the extensive 5' and 3' UTRs of bovine, human, pig, chick and rat link protein mRNA. As evidence that the UTRs might play a role in regulation of link protein mRNA turnover, multiple occurrences of the adenosine-uridine binding factor motif A(Ua)A were found to be conserved between species within 3' UTRs. A polyadenylation signal was conserved between the bovine and chicken sequence, use of which would result in the smallest of multiple bovine link protein mRNA species observed by Northern blot analysis.

Animals↗

PubMed1994

Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension.

To define the influence of time in culture on gene expression for extracellular matrix proteins we have examined the progression of changes in gene expression for chondrocyte extracellular matrix proteins from the time the chondrocytes are initially isolated from their native cartilaginous matrix through 13 days of high-density culture. We have also determined the effect of matrix depletion and shape change by enzymatically resuspending cells after 6 days in culture and sampling the replated cells at intervals up to 13 days. Northern blots of chondrocyte RNA were hybridized with probes for collagen alpha 1(II), alpha 1(I), aggrecan, link protein, and decorin mRNA. The steady-state level of alpha 1(II) collagen mRNA dropped to 45% of the initial value within 24 h, with a further decrease to 21% by Day 3. A similar decline occurred, but less rapidly in ascorbate supplemented cultures with values of 78%, at 24 h and 62% at Day 3. Very low levels of alpha 1(I) collagen mRNA were detectible in cells maintained for 2 weeks without ascorbate supplementation. Type I collagen alpha 1(I) mRNA was not detected in freshly isolated chondrocytes or at the earliest times in culture but was increasingly abundant from Days 5-13 in the presence of ascorbate. Ascorbic acid supplementation altered the pattern of aggrecan expression over time. Without ascorbate there was an increase in steady-state aggrecan mRNA with time in culture, but in the presence of ascorbate, aggrecan mRNA levels peaked at early culture times and progressively diminished. Decorin steady-state mRNA levels in cultures not supplemented with ascorbic acid steadily increased over time in culture following a lag of several days. In cultures treated with ascorbate, however, there was a progressive increase in decorin steady-state mRNA levels from the first day in culture. Resuspending chondrocytes by digestion of the cell layer with pronase and collagenase at Day 6, which resulted in a transient shape change as well as matrix depletion, resulted in a greater than 2-fold increase in alpha 1(II) mRNA at Day 7 in ascorbate supplemented cultures. Only with ascorbate was there a small increase in decorin mRNA at Day 7, after resuspension. Aggrecan mRNA, however, showed a 3-fold increase without ascorbate and a 10-fold increase with ascorbate within 24 h of resuspension. Similarly, link protein steady-state mRNA showed an 8-fold increase without ascorbate and a 9-fold increase with ascorbate within 24 h after resuspension.

Aggrecans↗