Stepwise construction of triple-helical heparin binding sites using peptide models

Doss-Pepe, Ellen; Deprez, Paola; Silva, Teresita; Inestrosa, Nibaldo C.; Kirkpatrick, Alan; Ramshaw, John A. M.; Brodsky, Barbara

doi:10.1016/j.bbapap.2003.11.034

Cited by 11 publications

(17 citation statements)

References 38 publications

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“…In addition, expression of a second construct, VCLH, which included a heparin binding domain, H, substituted using site directed mutagenesis into the CL sequence, was also tested. In this construct, a heparin binding sequence, GRPGKRGKQGQK, derived from the collagenous tail of acetylcholine esterase [23,24] was substituted via PCR directed integration into the CL sequence GPAGPMGPAGER that starts at base pair 564/amino acid residue 188 (Figure 1). The substitution was confirmed by DNA sequencing.…”

Section: Resultsmentioning

confidence: 99%

“…It can be modified to include additional copies of the collagen domain [19] or by inclusion of other mammalian collagen sequences [20] or other triple helical proteins. For example, it is possible to introduce specific functions, such as cell binding via a mammalian integrin binding site [21,22] or inclusion of a heparin binding domain [23,24] into the bacterial collagen domain.…”

Section: Introductionmentioning

confidence: 99%

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Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications

et al. 2012

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BackgroundCollagen has proved valuable as biomedical materials for a range of clinical applications, particularly in wound healing. It is normally produced from animal sources, such as from bovines, but concerns have emerged over transmission of diseases. Recombinant collagens would be preferable, but are difficult to produce. Recently, studies have shown that ‘collagens’ from bacteria, including Streptococcus pyogenes, can be produced in the laboratory as recombinant products, and that these are biocompatible. In the present study we have established that examples of bacterial collagens can be produced in a bioreactor with high yields providing proof of manufacture of this important group of proteins.ResultsProduction trials in shake flask cultures gave low yields of recombinant product, < 1 g/L. Increased yields, of around 1 g/L, were obtained when the shake flask process was transferred to a stirred tank bioreactor, and the yield was further enhanced to around 10 g/L by implementation of a high cell density fed-batch process and the use of suitably formulated fully defined media. Similar yields were obtained with 2 different constructs, one containing an introduced heparin binding domain. The best yields, of up to 19 g/L were obtained using this high cell density strategy, with an extended 24 h production time.ConclusionsThese data have shown that recombinant bacterial collagen from S. pyogenes, can be produced in sufficient yield by a scalable microbial production process to give commercially acceptable yields for broad use in biomedical applications.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications

et al. 2012

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“…Furthermore, the homotrimer has a triple-helical conformation, whereas HepV does not, as demonstrated by circular dichroism, and that might contribute to their different requirements for sulfate group binding. It has been previously reported for the heparin binding domains of the acetylcholinesterase collagen tail that the addition of increased amounts of heparin can increase the triple helix content and the thermal stability of triple-helical peptide models (40,41). However, the addition of heparin and heparan sulfate at different concentrations failed to induce significant structural change in HepV even during an overnight incubation.…”

Section: Inhibitionmentioning

confidence: 81%

Structural Requirements for Heparin/Heparan Sulfate Binding to Type V Collagen

Ricard‐Blum

Beraud

Raynal

et al. 2006

Journal of Biological Chemistry

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Collagen-proteoglycan interactions participate in the regand native collagen V molecules formed much more stable complexes with heparin than HepV, and collagen V bound to heparin/heparan sulfate with a higher affinity (in the nanomolar range) than HepV. Heat and chemical denaturation strongly decreased the binding, indicating that the triple helix plays a major role in stabilizing the interaction with heparin. Collagen V and HepV may play different roles in cell-matrix interactions and in matrix assembly or remodeling mediated by their specific interactions with heparan sulfate.Proteoglycans are widely distributed at the cell surface and in the extracellular matrix. They consist of a protein core to which one or more glycosaminoglycan side chains are covalently attached, conferring a strong negative charge on proteoglycans (1, 2). Interactions of proteoglycans with a number of matrix proteins, including collagens, are important in regulating cell behavior and fibril formation during development and pathophysiological events. Interactions with collagens can be dependent on the proteoglycan core protein or on the glycosaminoglycan chains. The heparan sulfate chain has various important biological properties and influences cell behavior through interactions with a variety of matrix proteins. Heparin binding sites have been identified and characterized in a wide range of matrix proteins, including collagens, but the structural requirements for heparan sulfate to bind these proteins are not fully understood.Collagen V, although a quantitatively minor component of connective tissues, plays a crucial role in matrix organization. Several isoforms of collagen V, including hybrid molecules containing collagen XI chains (3), occur in tissues, but the predominant molecular form is the heterotrimer (␣1(V)) 2 ␣2(V), whereas the ␣1(V)␣2(V)␣3(V) molecule and the (␣1(V)) 3 homotrimer are minor isoforms with a more restricted tissue distribution. Collagen V interacts with proteoglycans, which are widely distributed at the cell surface and in the extracellular matrix. It binds to the two small proteoglycans, decorin and biglycan, to the proteoglycan form of macrophage colony-stimulating factor (4), to the membrane chondroitin sulfate proteoglycan NG2 (5), and to syndecan-1 (6, 7). Binding to collagen V involves either the core protein or the glycosaminoglycan chains. Collagen V was shown to bind heparin through a 12-kDa fragment of the ␣1(V) chain referred to as HepV. Interestingly, we also showed that the recombinant HepV fragment supports heparin-dependent cell adhesion (8). The binding site for heparin is found in the ␣1 chain of collagen V, whereas the two other chains of collagen V, ␣2(V) and ␣3(V), do not bind to heparin. Sequence alignment of ␣ chains of collagens V and XI indicated that these two collagen types may use a common sequence motif to interact with heparin. This hypothesis was confirmed experimentally by others (9). The recombinant production of HepV in Escherichia coli has set the stage for identifying the hepa...

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“…The peptide chosen was (GPO) 2 GROGKRGKO(GPO) 3 GG, a peptide with a heparin binding motif that was previously reported to form a triple-helical structure with a T m of ~26.9°C 27 . Addition of this basic peptide to GNNQQNY (c=0.45mM) at 20°C in a 1:2 ratio increased the lag phase (from ~4 hrs to ~7 hrs), but this delay was less than seen for (POG) 10 .…”

Section: Resultsmentioning

confidence: 99%

“…The GNNQQNY and the hybrid peptides were synthesized by Tufts University Core Facility, Boston, MA. A peptide of sequence (GPO) 2 GROGKRGKO(GPO) 3 GG was synthesized as previously described 27 . A peptide with a sequence similar to (POG) 10 , but missing one Gly, (POG) 4 PO(POG) 5 , was synthesized previously and shown to be unable to form the triple helical structure 28 .…”

Section: Methodsmentioning

confidence: 99%

A peptide study of the relationship between the collagen triple‐helix and amyloid

et al. 2012

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Type XXV collagen, or Collagen-Like Amyloidogenic Component (CLAC), is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer’s disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro-Hyp-Gly)10; an amyloidogenic peptide GNNQQNY; and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)n domains. CD and NMR spectroscopy showed the GNNQQNY peptide formed a random coil structure, while the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple-helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, while amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple-helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple-helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly-Xaa-Yaa sequence and required the triple-helix conformation. The inhibitory effect of the collagen triple-helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation.

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Stepwise construction of triple-helical heparin binding sites using peptide models

Cited by 11 publications

References 38 publications

Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications

Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications

Structural Requirements for Heparin/Heparan Sulfate Binding to Type V Collagen

A peptide study of the relationship between the collagen triple‐helix and amyloid

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