Roles of aggrecan domains in biosynthesis, modification by glycosaminoglycans and product secretion

2001 ◽  
Vol 354 (1) ◽  
pp. 199-207 ◽  
Author(s):  
Chris KIANI ◽  
Vivian LEE ◽  
Liu CAO ◽  
Liwen CHEN ◽  
Yaojiong WU ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Unique Feature ◽  
Chondroitin Sulphate ◽  
Attachment Site ◽  
Structural Similarity ◽  
Globular Domain ◽  
Keratan Sulphate ◽  
Central Sequence ◽  
Domain Sequence ◽  
C Terminus

Aggrecan is a member of the chondroitin sulphate (CS) proteoglycan family, which also includes versican/PG-M, neurocan and brevican. Members of this family exhibit structural similarity: a G1 domain at the N-terminus and a G3 domain at the C-terminus, with a central sequence for modification by CS chains. A unique feature of aggrecan is the insertion of three additional domains, an inter-globular domain (IGD), a G2 domain and a keratan sulphate (KS) domain (sequence modified by KS chains), between the G1 domain and the CS domain (sequence modified by CS chains). The G1 and G3 domains have been implicated in product secretion, but G2, although structurally similar to the tandem repeats of G1, performs an unknown function. To define the functions of each aggrecan domain in product processing, we cloned and expressed these domains in various combinations in COS-7 cells. The results indicated that the G3 domain enhanced product secretion, alone or in combination with the KS or CS domain, and promoted glycosaminoglycan (GAG) chain attachment. Constructs containing the G1 domain were not secreted. Addition of a CS domain sequence to G1 reduced this inhibition, but GAG chain attachment was still decreased. The potential GAG chain attachment site in the IGD was occupied by GAGs, and IGD product was secreted efficiently. The KS domain was modified by GAG chains and secreted. Finally, the G2 domain was expressed but not secreted, and inhibited secretion of the IGD when expressed as an IGD–G2 combination.

scholarly journals Cell-type specific adhesive interactions of skeletal myoblasts with thrombospondin-1.

1994 ◽  
Vol 5 (4) ◽  
pp. 423-437 ◽  
Author(s):  
J C Adams ◽  
J Lawler
Keyword(s):  
Skeletal Muscle ◽  
Fusion Protein ◽  
Chondroitin Sulphate ◽  
Attachment Site ◽  
Globular Domain ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Chondroitin Sulphate Proteoglycans ◽  
Thrombospondin 1 ◽  
Carboxy Terminal

Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein that may play important roles in the morphogenesis and repair of skeletal muscle. To begin to explore the role of thrombospondin-1 in this tissue, we have examined the interactions of three rodent skeletal muscle cell lines, C2C12, G8, and H9c2, with platelet TSP-1. The cells secrete thrombospondin and incorporate it into the cell layer in a distribution distinct from that of fibronectin. Myoblasts attach and spread on fibronectin- or thrombospondin-coated substrates with similar time and concentration dependencies. Whereas cells adherent on fibronectin organize actin stress fibers, cells adherent on TSP-1 display prominent membrane ruffles and lamellae that contain radial actin microspikes. Attachment to thrombospondin-1 or the 140-kDa tryptic fragment is mediated by interactions with the type 1 repeats and the carboxy-terminal globular domain. Attachment is not inhibited by heparin, GRGDSP peptide, or VTCG peptide but is inhibited by chondroitin sulphate A. Integrins of the beta 1 or alpha V subgroups do not appear to be involved in myoblast attachment to TSP-1; instead, this process depends in part on cell surface chondroitin sulphate proteoglycans. Whereas the central 70-kDa chymotryptic fragment of TSP-1 does not support myoblast attachment, the carboxy-terminal domain of TSP-1 expressed as a fusion protein in the bacterial expression vector, pGEX, supported myoblast attachment to 30% the level of intact TSP-1. Thrombospondin-4 (TSP-4) is also present in skeletal muscle and a fusion protein containing the carboxy-terminal domain of TSP-4 also supported myoblast adhesion, although this protein was less active on a molar basis than the TSP-1 fusion protein. Thus, the carboxyterminal domain of TSP-1 appears to contain a primary attachment site for myoblasts, and this activity is present in a second member of the thrombospondin family.

scholarly journals Proteoglycans isolated from dissociative extracts of differently aged human articular cartilage: characterization of naturally occurring hyaluronan-binding fragments of aggrecan

1994 ◽  
Vol 304 (3) ◽  
pp. 887-894 ◽  
Author(s):  
V Vilim ◽  
A J Fosang
Keyword(s):  
Articular Cartilage ◽  
Density Gradient ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Proteolytic Processing ◽  
Human Articular Cartilage ◽  
Globular Domain ◽  
Keratan Sulphate ◽  
Core Proteins

Proteoglycans extracted with 4 M guanidinium chloride from young (mean 20 years) or old (mean 79 years) macroscopically normal human articular cartilage were separated by density gradient centrifugation and Q-Sepharose chromatography and characterized by gradient gel SDS/PAGE and immunodetection before and after removal of glycosaminoglycan chains. The extracts contained two large populations of aggrecan, a population of small N-terminal aggrecan fragments, as well as decorin, biglycan and fibromodulin. The distribution of all these species in density gradient fractions has been determined. The large aggrecan populations comprised four different chondroitin sulphate-bearing core proteins while the population of smaller fragments comprised eight different components. The two smallest fragments (35 and 42 kDa), identified as the first globular domain of aggrecan (N-terminal) (G1) and containing no glycosaminoglycan, were detected only in extracts of old cartilage. A 55 and a 70 kDa fragment of G1 were present in both keratan sulphate-containing and non-keratan sulphate-containing forms. Four other fragments, each containing keratan sulphate epitopes, were identified and these contained either G1 epitopes (one 95 kDa species), or G1 and G2 epitopes (three species). These results have suggested that proteolytic processing at the N-terminus is more extensive than has previously been recognized and raises the possibility that more than one proteinase may be involved in aggrecan degradation in vivo. With the exception of the two smallest G1 fragments, the repertoire of proteoglycan fragments found in young and old human articular cartilage is essentially the same, although the relative abudnance of various species differed. The older tissue contains a larger proportion of C-terminally truncated aggrecan fragments and a significantly decreased content of decorin and biglycan.

scholarly journals Mature bovine articular cartilage contains abundant aggrecan that is C-terminally truncated at Ala719-Ala720, a site which is readily cleaved by m-calpain

2004 ◽  
Vol 382 (1) ◽  
pp. 253-259 ◽  
Author(s):  
Hidefumi OSHITA ◽  
John D. SANDY ◽  
Kiichi SUZUKI ◽  
Atsushi AKAIKE ◽  
Yun BAI ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Globular Domain ◽  
Cleavage Sites ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
The Matrix ◽  
A Site

Extracts of normal mature articular cartilage contain aggrecan molecules which bear the G1 domain (the N-terminal globular domain of aggrecan) and are C-terminally truncated by proteolysis at a number of sites. A proportion of these molecules are generated by an aggrecanase and/or matrix-metalloproteinase-mediated cleavage in the IGD (interglobular domain between the G1 and G2 domains of aggrecan). However, the proteinase(s) responsible for formation of the majority of the larger G1-G2 and glycosaminoglycan-bearing truncated species is (are) unknown. N-terminal sequencing of aggrecan core fragments generated by m-calpain digestion of bovine aggrecan has identified four novel cleavage sites: one within the CS (chondroitin sulphate)-1 domain (at one or more of the bonds Ser1229–Val1230, Ser1249–Val1250, Ser1287–Val1288, Gly1307–Val1308 and Ser1346–Val1347), two within the IGD (at bonds Ala474–Ala475 and Gly365–Gly366) and one within the KS (keratan sulphate) domain (at Ala719–Ala720). A new monoclonal antibody (SK-28) to the C-terminal neoepitope at M710VTQVGPGVA719 showed that aggrecan products generated by this cleavage are present in high abundance in mature bovine articular cartilage extracts. We conclude that m-calpain, or an unidentified proteinase with the capacity to cleave at the same site, is active during aggrecan biosynthesis/secretion by mature chondrocytes or in the matrix of mature bovine articular cartilage in vivo.

Healing of Circular Defects in the Rabbit Medial Meniscus Can Occur Spontaneously and Is not Improved by Intra-Articular Hyaluronic Acid

1996 ◽  
Vol 09 (02) ◽  
pp. 60-5 ◽  
Author(s):  
N. Hope ◽  
P. Ghosh ◽  
S. Collier
Keyword(s):  
Hyaluronic Acid ◽  
Medial Meniscus ◽  
Chondroitin Sulphate ◽  
Rabbit Model ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Keratan Sulphate ◽  
Meniscal Healing ◽  
Meniscus Healing ◽  
Made In

SummaryThe aim of this study was to determine the effects of intra-articular hyaluronic acid on meniscal healing. Circular defects, 1.0 mm in diameter, were made in the anterior third of the medial meniscus in rabbits. In one joint, 0.4 ml hyaluronic acid (Healon®) was instilled, and in the contralateral (control) joint, 0.4 ml Ringer’s saline. Four rabbits were killed after four, eight and 12 weeks and the menisci examined histologically. By eight weeks most of the lesions had healed by filling with hyaline-like cartilage. Healing was not improved by hyaluronic acid treatment. The repair tissue stained strongly with alcian blue, and the presence of type II collagen, keratan sulphate, and chondroitin sulphate was demonstrated by immunohistochemical localisation. In contrast to the circular defects, longitudinal incisions made in the medial menisci of a further six rabbits did not show any healing after 12 weeks, indicating that the shape of the lesion largely determined the potential for healing.The effect of hyaluronic acid on meniscal healing was tested in a rabbit model. With one millimeter circular lesions in the medial meniscus, healing by filling with hyalinelike cartilage was not significantly affected by the application of hyaluronic acid intra-articularly at the time of surgery, compared to saline controls, as assessed histologically four, eight and 12 weeks after the operation.

scholarly journals Structural Analysis of the Partially Disordered Protein EspK from Mycobacterium tuberculosis

Crystals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 18
Author(s):  
Abril Gijsbers ◽  
Nuria Sánchez-Puig ◽  
Ye Gao ◽  
Peter J. Peters ◽  
Raimond B. G. Ravelli ◽  
...  
Keyword(s):  
Host Response ◽  
Limited Proteolysis ◽  
Maximal Dimension ◽  
Globular Domain ◽  
Saxs Data ◽  
X Ray ◽  
Disordered Protein ◽  
X Ray Scattering ◽  
C Terminus ◽  
New Treatments

For centuries, tuberculosis has been a worldwide burden for human health, and gaps in our understanding of its pathogenesis have hampered the development of new treatments. ESX-1 is a complex machinery responsible for the secretion of virulence factors that manipulate the host response. Despite the importance of these secreted proteins for pathogenicity, only a few of them have been structurally and functionally characterised. Here, we describe a structural study of the ESX-secretion associated protein K (EspK), a 74 kDa protein known to be essential for the secretion of other substrates and the cytolytic effects of ESX-1. Small-Angle X-ray Scattering (SAXS) data show that EspK is a long molecule with a maximal dimension of 228 Å. It consists of two independent folded regions at each end of the protein connected by a flexible unstructured region driving the protein to coexist as an ensemble of conformations. Limited proteolysis identified a 26 kDa globular domain at the C-terminus of the protein consisting of a mixture of α-helices and β-strands, as shown by circular dichroism (CD) and SAXS. In contrast, the N-terminal portion is mainly helical with an elongated shape. Sequence conservation suggests that this architecture is preserved amongst the different mycobacteria species, proposing specific roles for the N- and C-terminal domains assisted by the middle flexible linker.

Regulatory function of the Saccharomyces cerevisiae RAS C-terminus

1987 ◽  
Vol 7 (7) ◽  
pp. 2309-2315
Author(s):  
M S Marshall ◽  
J B Gibbs ◽  
E M Scolnick ◽  
I S Sigal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Adenylate Cyclase ◽  
Attachment Site ◽  
Hydrolytic Activity ◽  
Regulatory Function ◽  
Coding Region ◽  
Activating Mutations ◽  
Terminal Domain ◽  
C Terminus ◽  
Type Gene

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.

scholarly journals RepeatsDB in 2021: improved data and extended classification for protein tandem repeat structures

2020 ◽  
Vol 49 (D1) ◽  
pp. D452-D457
Author(s):  
Lisanna Paladin ◽  
Martina Bevilacqua ◽  
Sara Errigo ◽  
Damiano Piovesan ◽  
Ivan Mičetić ◽  
...  
Keyword(s):  
Protein Data Bank ◽  
Tandem Repeat ◽  
Tandem Repeats ◽  
Classification Scheme ◽  
Sequence Similarity ◽  
Protein Structures ◽  
Hierarchical Classification ◽  
Structural Similarity ◽  
Data Bank ◽  
Similarity Class

Abstract The RepeatsDB database (URL: https://repeatsdb.org/) provides annotations and classification for protein tandem repeat structures from the Protein Data Bank (PDB). Protein tandem repeats are ubiquitous in all branches of the tree of life. The accumulation of solved repeat structures provides new possibilities for classification and detection, but also increasing the need for annotation. Here we present RepeatsDB 3.0, which addresses these challenges and presents an extended classification scheme. The major conceptual change compared to the previous version is the hierarchical classification combining top levels based solely on structural similarity (Class > Topology > Fold) with two new levels (Clan > Family) requiring sequence similarity and describing repeat motifs in collaboration with Pfam. Data growth has been addressed with improved mechanisms for browsing the classification hierarchy. A new UniProt-centric view unifies the increasingly frequent annotation of structures from identical or similar sequences. This update of RepeatsDB aligns with our commitment to develop a resource that extracts, organizes and distributes specialized information on tandem repeat protein structures.

scholarly journals Twin-arginine translocation-arresting protein regions contact TatA and TatB

2014 ◽  
Vol 395 (7-8) ◽  
pp. 827-836 ◽  
Author(s):  
Johannes Taubert ◽  
Thomas Brüser
Keyword(s):  
Signal Peptide ◽  
Sequence Motif ◽  
Globular Domain ◽  
Mature Protein ◽  
Linker Region ◽  
Twin Arginine Translocation ◽  
C Terminus ◽  
Translocation Efficiency ◽  
Folded Proteins

Abstract Tat systems translocate folded proteins across biological membranes of prokaryotes and plant plastids. TatBC complexes recognize N-terminal Tat signal peptides that contain a sequence motif with two conserved arginines (RR-motif), and transport takes place after a recruitment of TatA. Unfolded Tat substrate domains lower translocation efficiency and too long linkers lead to translocation arrest. To identify the components that interact with transported proteins during their passage through the translocon, we used a Tat substrate that arrests translocation at a long unfolded linker region, and we chose in vivo site-directed photo cross-linking to specifically detect the interactions of this linker region. For comparison, we included the interactions of the signal peptide and of the folded domain at the C-terminus of this construct. The data show that the linker contacts only two, structurally similar Tat components, namely TatA and TatB. These contacts depend on the recognition of the Tat-specific signal peptide. Only when membrane translocation of the globular domain was allowed – i.e., in the absence of the linker – we observed the same TatAB-contacts also to the globular domain. The data thus suggest that mature protein domains are translocated through a TatAB environment.

Association of a Variable Number of Tandem Repeats (VNTR) in Glycoprotein Ibα and HPA-2 Alloantigens

1994 ◽  
Vol 72 (05) ◽  
pp. 757-761 ◽  
Author(s):  
S Simsek ◽  
P M M Bleeker ◽  
C E van der Schoot ◽  
A E G Kr von dem Borne
Keyword(s):  
Amino Acid ◽  
Tandem Repeats ◽  
Mononuclear Cells ◽  
Human Platelet ◽  
Strong Association ◽  
Variable Number ◽  
Platelet Glycoprotein ◽  
Globular Domain ◽  
Vntr Polymorphism ◽  
Pcr Products

SummaryThe human platelet alloantigen HPA-2 (Koa/Kob) system is involved in two clinical syndromes, neonatal alloimmune thrombocytopenia and platelet transfusion refractoriness. Wb have previously described that the human platelet alloantigens HPA-2a(Kob) and HPA-2b(Koa), are caused by a Thrl45Met amino acid polymorphism in the N-terminal globular domain of the human platelet glycoprotein (GP) Ibα. In the present study the question was addressed as to whether a genetic association exists between this Thrl45Met polymorphism and the recently described variable number of tandem repeat (VNTR) polymorphism in GP Ibα. Such an association has already been suggested by serological analysis (Ishida et al., 1991). This VNTR polymorphism results from a 13-amino-acid sequence repeat in the macroglycopeptide region of GP Ibα. Therefore, we developed a PCR method to analyze the VNTR region of 106 normal individuals who were also analyzed for the HPA-2 polymorphism. In this method genomic DNA derived from mononuclear cells was purified, the polymorphic region was amplified by PCR and was electrophoresed on agarose gels. Differences in the size of the PCR products made VNTR typing possible. Genotyping for the HPA-2 system was done by allele-specific restriction site analysis of PCR products with the restriction enzyme Sfa NI. The DNA derived from 12 HPA-2 (a-b+) subjects, contained only the B variant (with 3 repeats) of the VNTR polymorphism. The D variant (with 1 repeat) was only found in HPA-2a positive individuals. The C variant (with 2 repeats) was found to be strongly associated with HPA-2a. However, two members of a family with a HPA-2 (a+b+) genotype were found to be homozygous for the C variant of the VNTR polymorphism. This shows that the C variant can also be associated with HPA-2b. The A variant (with 4 repeats) was not encountered in the population studied. The strong association of HPA-2 and VNTR polymorphism, lying 761 bp apart on the GP Ibα gene, indicates linkage disequilibrium.

scholarly journals Studies on protein–polysaccharides from pig laryngeal cartilage. Extraction and purification

1969 ◽  
Vol 113 (5) ◽  
pp. 879-884 ◽  
Author(s):  
C. P. Tsiganos ◽  
Helen Muir
Keyword(s):  
Hyaluronic Acid ◽  
Chloride Solution ◽  
Sodium Acetate ◽  
Serum Proteins ◽  
Chondroitin Sulphate ◽  
Calcium Chloride Solution ◽  
Keratan Sulphate ◽  
Laryngeal Cartilage ◽  
Extraction And Purification ◽  
Degradative Enzymes

1. Protein–polysaccharides of chondroitin sulphate were extracted from fresh laryngeal cartilage at pH6·8 by two procedures. Procedure I consisted of brief low-speed homogenization in 0·15m (iso-osmotic) sodium acetate and procedure II consisted of longer homogenization followed by prolonged extraction in 10% calcium chloride solution. 2. The protein–polysaccharides in both extracts were isolated and purified by precipitation with 9-aminoacridine hydrochloride. They were free from serum proteins, collagen and nucleic acids and also of degradative enzymes. The absence of such enzymes was shown by viscosity measurements on solutions of protein–polysaccharides incubated for up to 24hr. at pH4 and 6·8. 3. Mannose, glucose or fucose were not detected by paper chromatography and only traces of sialic acid were present. 4. The yield with procedure II was twice that with procedure I and the products differed in their protein and glucosamine contents. 5. Hyaluronic acid was unlikely to have been precipitated at an acid pH, so the glucosamine was attributed to keratan sulphate, as serum proteins were absent. There was no free keratan sulphate in the preparation. 6. Both preparations were heterogeneous in the ultracentrifuge, showing at least three components.

Sign in / Sign up

Export Citation Format

Share Document