Decorin binds fibrinogen in a Zn2+-dependent interaction

We have previously shown that decorin, a member of the small leucine-rich proteoglycan family of extracellular matrix proteoglycans/glycoproteins is a Zn(2+) metalloprotein at physiological Zn(2+) concentrations (Yang, V. W-C., LaBrenz, S. R., Rosenberg, L. C., McQuillan, D., and H??k, M. (1999) J. Biol. Chem. 274, 12454-12460). We now report that the decorin proteoglycan binds fibrinogen in the presence of Zn(2+). The fibrinogen-binding site is located in the N-terminal domain of the decorin core protein and a 45-amino acid peptide representing this domain binds to the fibrinogen D fragment with an apparent K(D) of 1.7 x 10(-6) m, as determined from fluorescence polarization data. Furthermore, we show that Zn(2+) promotes the self-association of decorin. The N-terminal domain of the core protein also mediates this activity. The results of solid-phase binding assays and gel filtration chromatography suggest that the N-terminal domain of decorin, when present at low micromolar concentrations, forms an oligomer in a Zn(2+)-dependent manner. Thus, Zn(2+) appears to play a pivotal role in the interactions and biological function of decorin.     

Structural correlations in the family of small leucine-rich repeat proteins and proteoglycans

The family of small leucine-rich repeat proteins and proteoglycans (SLRPs) contains several extracellular matrix molecules that are structurally related by a protein core composed of leucine-rich repeats (LRRs) flanked by two conserved cysteine-rich regions. The small proteoglycan decorin is the archetypal SLRP. Decorin is present in a variety of connective tissues, typically "decorating" collagen fibrils, and is involved in important biological functions, including the regulation of the assembly of fibrillar collagens and modulation of cell adhesion. Several SLRPs are known to regulate collagen fibrillogenesis and there is evidence that they may share other biological functions. We have recently determined the crystal structure of the protein core of decorin, the first such determination of a member of the SLRP family. This structure has highlighted several correlations: (1) SLRPs have similar internal repeat structures; (2) SLRP molecules are far less curved than an early model of decorin based on the three-dimensional structure of ribonuclease inhibitor; (3) the N-terminal and C-terminal cysteine-rich regions are conserved capping motifs. Furthermore, the structure shows that decorin dimerizes through the concave surface of its LRR domain, which has been implicated previously in its interaction with collagen. We have established that both decorin and opticin, another SLRP, form stable dimers in solution. Conservation of residues involved in decorin dimerization suggests that the mode of dimerization for other SLRPs will be similar. Taken together these results suggest the need for reevaluation of currently accepted models of SLRP interaction with their ligands.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Molecular Cloning of Testican-2

We have screened a human cDNA library using an expressed sequence tag related to the BM-40/secreted protein, acidic and rich in cysteine (SPARC)/osteonectin family of proteins and isolated a novel cDNA. It encodes a protein precursor of 424 amino acids that consists of a signal peptide, a follistatin-like domain, a Ca2+-binding domain, a thyroglobulin-like domain, and a C-terminal region with two putative glycosaminoglycan attachment sites. The protein is homologous to testican-1 and was termed testican-2. Testican-1 is a proteoglycan originally isolated from human seminal plasma that is also expressed in brain. Northern blot hybridization of testican-2 showed a 6.1-kb mRNA expressed mainly in CNS but also found in lung and testis. A widespread expression in multiple neuronal cell types in olfactory bulb, cerebral cortex, thalamus, hippocampus, cerebellum, and medulla was detected by in situ hybridization. A recombinant fragment consisting of the Ca2+-binding EF-hand domain and the thyroglobulin-like domain of testican-2 showed a reversible Ca2+-dependent conformational change in circular dichroism studies. Testican-1 and -2 form a novel Ca2+-binding proteoglycan family built of modular domains with the potential to participate in diverse steps of neurogenesis.     

Identification and characterization of a novel interaction between pulmonary surfactant protein D and decorin

Surfactant-associated protein D (SP-D) is a collectin that is present in lung surfactant and mucosal surfaces. Although SP-D regulates diverse functions, only a few proteins are known to bind to this collectin. Here we describe the co-purification of decorin, a novel SP-D-binding protein, from amniotic fluid. The human decorin that co-purified with SP-D is a 130-150-kDa proteoglycan, which has a 46-kDa protein core and approximately 90-kDa dermatan sulfate chain. Both native and recombinant decorin can bind to SP-D that is already bound to maltose-agarose matrix, and these SP-D-decorin complexes are dissociated at high salt (0.5-1.0 m NaCl) conditions, releasing the decorin. We further show that SP-D and decorin interact with each other (kd = 4 nm) by two mechanisms. First, the direct binding and competition experiments show that the carbohydrate recognition domain (CRD) of SP-D binds in a calcium dependent-manner to the sulfated N-acetyl galactosamine moiety of the glycosaminoglycan chain. Second, complement component C1q, a complement protein that is known to interact with decorin core protein via its collagen-like region, partially blocks the interaction between decorin and native SP-D. This protein, however, does not block the interaction between decorin and SP-D(n/CRD), a recombinant fragment that lacks the N-terminal and collagen-like regions. Furthermore, the core protein, obtained by chondroitin ABC lyase treatment of decorin, binds SP-D, but not SP-D(n/CRD). These findings suggest that decorin core protein binds the collagen-like region of the SP-D. Concentrations of decorin and SP-D are negatively correlated to each other, in amniotic fluid, implying a functional relevance for SP-D-decorin interaction, in vivo. Collectively, our results show that carbohydrate recognition domains of SP-D interact with the dermatan sulfate moiety of decorin via lectin activity and that the core protein of decorin binds the collagen-like region of SP-D in vitro, and these interactions may be operative in vivo.     

Binding of the proteoglycan decorin to collagen type VI.

We have examined the interactions between the small dermatan sulfate proteoglycan decorin and collagen types I-VI using solid phase binding assays. The results of these studies showed that 125I-decorin bound most efficiently to collagen type VI in a time- and concentration-dependent manner. Furthermore, this interaction was specific and of moderately high affinity (Kd approximately 3 x 10(-7) M). Binding of decorin to collagen type VI appears to involve the decorin core protein rather than the glycosaminoglycan side chains, since the isolated core protein as well as a recombinant fusion protein containing a major segment (65%) of the human decorin core protein inhibited binding of 125I-decorin to collagen type VI. Other related proteoglycans and their respective core proteins also inhibited the binding of 125I-decorin to collagen type VI, whereas unrelated proteins and isolated glycosaminoglycan chains were without effect. In addition to decorin, collagen type II was also shown to bind to immobilized collagen type VI. Both interactions were effectively inhibited by preincubation of the immobilized collagen VI with decorin or collagen type II. These results suggested that the collagen type VI molecule has binding sites for collagen type II and decorin which are located in close proximity on the collagen type VI molecule. Possible functional roles of these interactions are discussed.     

Interactions between thrombospondin and the small proteoglycan decorin: interference with cell attachment.

Decorin, a ubiquitous small interstitial dermatan sulfate proteoglycan, interacts with several extracellular matrix components, e.g., with type I collagen and fibronectin. Using a solid phase assay it is shown that the intact proteoglycan as well as its glycosaminoglycan-free core protein exhibits with KD values of about 5 nM and 2 nM, respectively, high affinity binding also to thrombospondin. However, the polysaccharide chain was required for an interaction with Sepharose-bound thrombospondin and served itself as ligand. In light of the results of binding studies with an N-terminal heparin-binding fragment of thrombospondin it is concluded that several structural features of thrombospondin and of decorin contribute to the mutual interaction of the two macromolecules. Thrombospondin substrata allowed attachment but prevented spreading of human skin fibroblasts. The addition of decorin or of its glycosaminoglycan-free core protein led to a considerable delay of cell attachment on a thrombospondin substrate. The strength of cell attachment appeared to be reduced. These data support the antiadhesive role of decorin regardless of whether subsequent cell spreading is supported or not.     

Apoptosis-linked gene product ALG-2 is a new member of the calpain small subunit subfamily of Ca2+-binding proteins.

ALG-2 is a newly discovered Ca2+-binding protein which has been demonstrated to be directly linked to apoptosis. Structurally, ALG-2 is expressed as a single polypeptide chain corresponding to a 22 kDa protein containing five putative EF-hand Ca2+-binding sites. In this work, we have developed an efficient expression and purification scheme for recombinant ALG-2. Utilizing this protocol, we can routinely obtain purified recombinant protein with a yield of approximately 100 mg per liter of bacterial cell cultures. Gel filtration and chemical cross-linking experiments have shown that Ca2+-free ALG-2 forms a weak homodimer in solution. Biochemical and spectroscopic studies of truncated and point mutants of ALG-2 demonstrated that the fifth EF-hand Ca2+-binding motif is likely to participate in the formation of the dimer complex. Experimentally, both the amino- and carboxyl-terminal truncated mutants of ALG-2 have shown their ability to retain the structural, as well as, Ca2+-binding integrity when individually expressed in bacteria. In this respect, the N-terminal domain encompasses the first two EF-hands, and the C-terminal domain contains the remaining three EF-hands. Combining mutagenesis and spectroscopic studies, we showed that ALG-2 possesses two strong Ca2+-binding sites. Employing fluorescence spectroscopy and circular dichroism, we showed that the binding of Ca2+ to ALG-2 induced significant conformational changes in both the N-terminal and C-terminal domains of the protein. Furthermore, our studies demonstrated that Ca2+ binding to both strong Ca2+-binding sites of ALG-2 is required for ion-induced aggregation of the protein. We also report here the expression, purification, and partial characterization of a Ca2+-binding-deficient ALG-2 mutant (Glu47Ala/Glu114Ala). In light of its much decreased affinity for Ca2+, this mutant could prove to be instrumental in elucidating the Ca2+-mediated function of ALG-2 within the context of its cellular environment.     

Collagen phagocytosis by fibroblasts is regulated by decorin

Decorin is a small, leucine-rich proteoglycan that binds to collagen and regulates fibrillogenesis. We hypothesized that decorin binding to collagen inhibits phagocytosis of collagen fibrils. To determine the effects of decorin on collagen degradation, we analyzed phagocytosis of collagen and collagen/decorin-coated fluorescent beads by Rat-2 and gingival fibroblasts. Collagen beads bound to gingival cells by alpha2beta1 integrins. Binding and internalization of decorin/collagen-coated beads decreased dose-dependently with increasing decorin concentration (p < 0.001). Inhibition of binding was sustained over 5 h (p < 0.001) and was attributed to interactions between decorin and collagen and not to decorin-collagen receptor interactions. Both the non-glycosylated decorin core protein and the thermally denatured decorin significantly inhibited collagen bead binding (approximately 50 and 89%, respectively; p < 0.05). Mimetic peptides corresponding to leucine-rich repeats 1-3, encompassed by a collagen-binding approximately 11-kDa cyanogen bromide fragment of decorin and leucine-rich repeats 4 and 5, previously shown to bind to collagen, were tested for their ability to inhibit collagen bead binding. Although the synthetic peptide 3 alone exhibited saturable binding to collagen, neither peptides 3 nor 1 and 2 markedly inhibited phagocytosis. Leucine-rich repeat 3 bound to a triple helical peptide containing the alpha2 integrin-binding site of collagen. When collagen beads were co-incubated with peptides 3 and 4, inhibition of collagen phagocytosis (55%) was equivalent to intact native/recombinant core protein. Thus a novel collagen binding domain in decorin acts cooperatively with leucine-rich repeat 4 to mask the alpha2beta1 integrin-binding site on collagen, an important sequence for the phagocytosis of collagen fibrils.     

Structural independence of the two EF-hand domains of caltractin

Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli. Ca2+ binding affinities and the Ca2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays. Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca2+-dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain. Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 microm) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals but is activated by the binding of a single ion.     

The macromolecular characteristics of cartilage proteoglycans do not change when synthesis is up-regulated by link protein peptide.

Previous studies have shown that a synthetic, unglycosylated analogue of the N-terminal peptide from link protein can function as a growth factor and up-regulate proteoglycan biosynthesis in explant cultures of normal human articular cartilage from a wide age range of subjects (McKenna et al., Arthritis Rheum. 41 (1998) 157-162). The present work further shows that link peptide increased proteoglycan synthesis by cartilage cultured in both the presence and absence of serum, suggesting that the mechanism of up-regulation may be different from that of insulin-like growth factors. The proteoglycans synthesised during stimulation with link peptide were of normal hydrodynamic size and the ratio of core protein to glycosaminoglycan side chains and the proportions of the large proteoglycan aggrecan to the small proteoglycans, decorin and biglycan, remained constant. Aggrecan molecules were equally capable of forming aggregates as those from control tissues and the relative proportions of decorin and biglycan were unchanged showing that both were co-ordinately up-regulated. These results confirmed that this novel peptide is a potent stimulator of proteoglycan synthesis by articular cartilage and showed that the newly synthesised proteoglycans were of normal composition.     

Derivation of Inhibitory Peptides to Target the Cardiac Troponin C–I Interaction as Potential Therapeutics for Heart Failure

The Ca²⁺-sensitive cardiac troponin (cTn) is a hetero-trimer complex consisting of three subunits cTnC, cTnI, and cTnT, which has been recognized as an important biomarker and a potential target of cardiovascular diseases. Previously, several small-molecule agents such as levosimendan and pimobendan have been successfully developed to target this protein for the treatment of heart failure. Here, instead of small-molecule chemical drugs, we purposed rational derivation of self-inhibitory peptides as potential biologic disruptors of cTnC–cTnI interaction from the interaction complex interface. In the procedure, the crystal structure of cTn trimer was examined in detail using bioinformatics approach, from which a peptide-mediated interaction between the N-terminal domain of cTnC and the switch region of cTnI was identified. The switch is a 19-mer peptide segment Swt that contains a structured helical core capped by a short N-terminal tripeptide and a disordered C-terminal tail. Structural and energetic analysis revealed that the Swt peptide binds independently to cTnC N-terminal domain, which can be stripped from the intact cTnI subunit to interact effectively with cTnC. Further investigations found that truncation of two N-terminal residues and five C-terminal residues of the full-length Swt peptide, resulting in a shortened version namely Swt-ΔN2ΔC5 peptide, would not cause substantial loss in its binding potency to cTnC. The computational finding was then confirmed by using fluorescence-based affinity assays; the Swt and Swt-ΔN2ΔC5 peptides was experimentally measured to have a moderately high affinity to the recombinant protein of human cTnC N-terminal domain with K _d values at micromolar level. The Swt and Swt-ΔN2ΔC5 are considered as inhibitory peptides that can be further optimized and modified to obtain high-affinity disruptors of cTnI–cTnC interaction.     

Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding

Protein engineering was used previously to convert maltose-binding protein (MBP) into a zinc biosensor. Zn(2+) binding by the engineered MBP was thought to require a large conformational change from "open" to "closed", similar to that observed when maltose is bound by the wild-type protein. We show that although this re-designed MBP molecule binds Zn(2+) with high affinity as previously reported, it does not adopt a closed conformation in solution as assessed by small-angle X-ray scattering. High-resolution crystallographic studies of the engineered Zn(2+)-binding MBP molecule demonstrate that Zn(2+) is coordinated by residues on the N-terminal lobe only, and therefore Zn(2+) binding does not require the protein to adopt a fully closed conformation. Additional crystallographic studies indicate that this unexpected Zn(2+) binding site can also coordinate Cu(2+) and Ni(2+) with only subtle changes in the overall conformation of the protein. This work illustrates that the energetic barrier to domain closure, which normally functions to maintain MBP in an open concentration in the absence of ligand, is not easily overcome by protein design. A comparison to the mechanism of maltose-induced domain rearrangement is discussed.     

Neutrophil recognition requires a Ca(2+)-induced conformational change in the lectin domain of GMP-140.

GMP-140, a receptor for myeloid cells that is expressed on surfaces of thrombin-activated platelets and endothelial cells, is a member of the selectin family of adhesion molecules that regulate leukocyte interactions with the blood vessel wall. Each selectin contains an N-terminal domain homologous to Ca(2+)-dependent lectins and mediates cell-cell contact by binding to oligosaccharide ligands in a Ca(2+)-dependent manner. The mechanisms by which Ca2+ promotes selectin-dependent cellular interactions have not been defined. We demonstrate that purified GMP-140 contains two high affinity binding sites for Ca2+ as measured by equilibrium dialysis (Kd = 22 +/- 2 microM). Occupancy of these sites by Ca2+ alters the conformation of the protein as detected by a reduction in intrinsic fluorescence emission intensity (Kd = 4.8 +/- 0.2 microM). This Ca(2+)-dependent conformational change exposes an epitope spanning residues 19-34 of the lectin domain that is recognized by a monoclonal antibody capable of blocking neutrophil adhesion to GMP-140 (half-maximal antibody binding at approximately 20 microM Ca2+). Furthermore, a synthetic peptide encoding this epitope, CQNRYTDLVAIQNKNE, inhibits neutrophil binding to GMP-140. Mg2+ also alters the conformation of the protein, but not in a manner that will support leukocyte recognition in the absence of Ca2+. There is a strong correlation between the Ca2+ levels required for neutrophil adhesion to GMP-140, for occupancy of the two Ca(2+)-binding sites, for the fluorescence-detected conformational change, and for exposure of the antibody epitope in the lectin domain. We conclude that binding of Ca2+ to high affinity sites on GMP-140 modulates the conformation of the lectin domain in a manner that is essential for leukocyte recognition.     

Distinct secondary structures of the leucine-rich repeat proteoglycans decorin and biglycan. Glycosylation-dependent conformational stability

Biglycan and decorin have been overexpressed in eukaryotic cells and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans (Hocking, A. M., Strugnell, R. A., Ramamurthy, P., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19571-19577; Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19578-19584). Far-UV CD spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the final form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that in this specific domain the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provide further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1 and 2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1 and 6 M urea, probably indicating the presence of stable unfolding intermediates.     

Multiple domains of the large fibroblast proteoglycan, versican.

The primary structure of a large chondroitin sulfate proteoglycan expressed by human fibroblasts has been determined. Overlapping cDNA clones code for the entire 2389 amino acid long core protein and the 20-residue signal peptide. The sequence predicts a potential hyaluronic acid-binding domain in the amino-terminal portion. This domain contains sequences virtually identical to partial peptide sequences from a glial hyaluronate-binding protein. Putative glycosaminoglycan attachment sites are located in the middle of the protein. The carboxy-terminal portion includes two epidermal growth factor (EGF)-like repeats, a lectin-like sequence and a complement regulatory protein-like domain. The same set of binding elements has also been identified in a new class of cell adhesion molecules. Amino- and carboxy-terminal portions of the fibroblast core protein are closely related to the core protein of a large chondroitin sulfate proteoglycan of chondrosarcoma cells. However, the glycosaminoglycan attachment regions in the middle of the core proteins are different and only the fibroblast core protein contains EGF-like repeats. Based on the similarities of its domains with various binding elements of other proteins, we suggest that the large fibroblast proteoglycan, herein referred to as versican, may function in cell recognition, possibly by connecting extracellular matrix components and cell surface glycoproteins.     

Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins

Normal and pathological turnover of proteoglycans in articular cartilage involves its cleavage close to the N-terminal G1 domain responsible for aggregation. A fragment containing G1 and G2 N-terminal domains of pig cartilage proteoglycans was therefore used as a substrate to investigate its degradation by the metalloproteinase stromelysin and related recombinant stromelysin enzymes. The stromelysins produced an apparent single cleavage yielding a G1 fragment of 56 kDa and a G2 fragment of 110 kDa. Rabbit bone stromelysin was much more active against the G1-G2 fragment and against proteoglycan aggregates than recombinant human stromelysin-1 and stromelysin-2. All metalloproteinase preparations were active against proteoglycan and the G1-G2 fragment at acid (pH 5.5) and neutral pH (7.4). N-terminal sequencing of the G2 fragment derived from the action of recombinant human stromelysin-1 revealed that cleavage between G1 and G2 occurred at the N-terminal end of the interglobular domain, close to the last cysteine in G1. The specific cleavage site was between an asparagine and a pair of phenylalanine residues, where the asparagine corresponds to residue 341 in human and rat mature core protein sequence.     

Sequence Analysis and Domain Motifs in the Porcine Skin Decorin Glycosaminoglycan Chain

Decorin proteoglycan is comprised of a core protein containing a single O-linked dermatan sulfate/chondroitin sulfate glycosaminoglycan (GAG) chain. Although the sequence of the decorin core protein is determined by the gene encoding its structure, the structure of its GAG chain is determined in the Golgi. The recent application of modern MS to bikunin, a far simpler chondroitin sulfate proteoglycans, suggests that it has a single or small number of defined sequences. On this basis, a similar approach to sequence the decorin of porcine skin much larger and more structurally complex dermatan sulfate/chondroitin sulfate GAG chain was undertaken. This approach resulted in information on the consistency/variability of its linkage region at the reducing end of the GAG chain, its iduronic acid-rich domain, glucuronic acid-rich domain, and non-reducing end. A general motif for the porcine skin decorin GAG chain was established. A single small decorin GAG chain was sequenced using MS/MS analysis. The data obtained in the study suggest that the decorin GAG chain has a small or a limited number of sequences.     

The primary structure of a new Mr 18,000 calcium vector protein from amphioxus

The amino acid sequence of a new Ca2+-binding protein (CaVP) from Amphioxus muscle (Cox, J. A., J. Biol. Chem. 261, 13173-13178) has been determined. The protein contains 161 amino acid residues and has a molecular weight of 18,267. The N terminus is blocked by an acetyl group. The two functional Ca2+-binding sites have been localized based on homology with known Ca2+-binding domains, on internal homology and on secondary structure prediction, and appear to be the domains III and IV. The C-terminal half of CaVP, which contains the two Ca2+-binding sites, shows a remarkable similarity with human brain calmodulin (45%) and with rabbit skeletal troponin C (40%). Functional domain III contains 2 epsilon-N-trimethyllysine residues in the alpha-helices flanking the Ca2+-binding loop. Sequence determination revealed two abortive Ca2+-binding domains in the N-terminal half of CaVP with a similarity of 24 and 30% as compared with calmodulin and troponin C, respectively. This half is also characterized by the presence of a disulfide bridge linking the N-terminal helix of domain I to the C-terminal helix of domain II. This disulfide bond is very resistant to reduction in the native state, but not in denatured CaVP. The optically interesting aromatic chromophores (2 tryptophan and 1 tyrosine residues) are all located in the nonfunctional domain II.