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Structural and functional investigations of Matrilin-1 A-domains reveal insights into their role in cartilage ECM assembly.

Fresquet M, Jowitt TA, Stephen LA, Ylöstalo J, Briggs MD - J. Biol. Chem. (2010)

Bottom Line: Initial characterization revealed that N-glycosylation did not affect secretion of these two proteins, nor did it alter their folding characteristics.However, removal of the glycosylation decreased their thermal stability.We then compared the effect of different cations on binding between both M1A domains and type II and IX collagens and showed that Zn(2+) also supports their interactions.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester M13 9PT, United Kingdom.

ABSTRACT
Matrilin-1 is expressed predominantly in cartilage and co-localizes with matrilin-3 with which it can form hetero-oligomers. We recently described novel structural and functional features of the matrilin-3 A-domain (M3A) and demonstrated that it bound with high affinity to type II and IX collagens. Interactions preferentially occurred in the presence of Zn(2+) suggesting that matrilin-3 has acquired a requirement for specific metal ions for activation and/or molecular associations. To understand the interdependence of matrilin-1/-3 hetero-oligomers in extracellular matrix (ECM) interactions, we have extended these studies to include the two matrilin-1 A-domains (i.e. M1A1 and M1A2 respectively). In this study we have identified new characteristics of the matrilin-1 A-domains by describing their glycosylation state and the effect of N-glycan chains on their structure, thermal stability, and protein-protein interactions. Initial characterization revealed that N-glycosylation did not affect secretion of these two proteins, nor did it alter their folding characteristics. However, removal of the glycosylation decreased their thermal stability. We then compared the effect of different cations on binding between both M1A domains and type II and IX collagens and showed that Zn(2+) also supports their interactions. Finally, we have demonstrated that both M1A1 domains and biglycan are essential for the association of the type II·VI collagen complex. We predict that a potential role of the matrilin-1/-3 hetero-oligomer might be to increase multivalency, and therefore the ability to connect various ECM components. Differing affinities could act to regulate the integrated network, thus coordinating the organization of the macromolecular structures in the cartilage ECM.

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The effect of glycosylation of matrilin-1 A-domains on the binding to type II and IX collagen measured using QCM-D. A, kinetics analysis of the binding between immobilized type II collagen and matrilin-3 and -1 A-domains in the presence of 1 mm Zn2+. B, normalized frequency shift (mass change) and dissipation shift versus time, detected during the association of matrilin-1 and -3 A-domains onto immobilized type II collagen (left panel). The right panel represents normalized changes in ΔD as a function of Δf for data shown in the left panel. C, normalized frequency change and dissipation change versus time, detected during the association of matrilin-3 and -1 A-domains onto immobilized type IX collagen (left panel). The right panel shows the D-f plot using QCM-D raw data at the third overtone (n = 3).
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Figure 5: The effect of glycosylation of matrilin-1 A-domains on the binding to type II and IX collagen measured using QCM-D. A, kinetics analysis of the binding between immobilized type II collagen and matrilin-3 and -1 A-domains in the presence of 1 mm Zn2+. B, normalized frequency shift (mass change) and dissipation shift versus time, detected during the association of matrilin-1 and -3 A-domains onto immobilized type II collagen (left panel). The right panel represents normalized changes in ΔD as a function of Δf for data shown in the left panel. C, normalized frequency change and dissipation change versus time, detected during the association of matrilin-3 and -1 A-domains onto immobilized type IX collagen (left panel). The right panel shows the D-f plot using QCM-D raw data at the third overtone (n = 3).

Mentions: The M1A2 domain showed the strongest affinity with a KD of ∼50 nm compared with M3A (∼91 nm) and M1A1 (∼104 nm) (Fig. 5A). This trend was in agreement with the calculated KD as determined by SPR analyses, which were 2.13 nm for M1A2, 6.2 nm for M3A, and 8.8 nm for M1A1 (Fig. 4B, top panel, and table). The difference in binding between M1A2 and M3A was also statistically significant (p < 0.001). However, no difference was detected between wild type M1A2 and the unglycosylated form (probably due to the fact that wild type M1A2 contains a high proportion of unglycosylated protein resulting in a masking effect). An interesting finding was that the unglycosylated form of M1A1 bound more strongly to type II collagen than the wild type molecule. This suggests that the flexible N-glycan chains might partially hide the binding site for type II collagen, thus narrowing the location of the binding site for this molecule to the opposite face of the MIDAS motif (Fig. 2D).


Structural and functional investigations of Matrilin-1 A-domains reveal insights into their role in cartilage ECM assembly.

Fresquet M, Jowitt TA, Stephen LA, Ylöstalo J, Briggs MD - J. Biol. Chem. (2010)

The effect of glycosylation of matrilin-1 A-domains on the binding to type II and IX collagen measured using QCM-D. A, kinetics analysis of the binding between immobilized type II collagen and matrilin-3 and -1 A-domains in the presence of 1 mm Zn2+. B, normalized frequency shift (mass change) and dissipation shift versus time, detected during the association of matrilin-1 and -3 A-domains onto immobilized type II collagen (left panel). The right panel represents normalized changes in ΔD as a function of Δf for data shown in the left panel. C, normalized frequency change and dissipation change versus time, detected during the association of matrilin-3 and -1 A-domains onto immobilized type IX collagen (left panel). The right panel shows the D-f plot using QCM-D raw data at the third overtone (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2962504&req=5

Figure 5: The effect of glycosylation of matrilin-1 A-domains on the binding to type II and IX collagen measured using QCM-D. A, kinetics analysis of the binding between immobilized type II collagen and matrilin-3 and -1 A-domains in the presence of 1 mm Zn2+. B, normalized frequency shift (mass change) and dissipation shift versus time, detected during the association of matrilin-1 and -3 A-domains onto immobilized type II collagen (left panel). The right panel represents normalized changes in ΔD as a function of Δf for data shown in the left panel. C, normalized frequency change and dissipation change versus time, detected during the association of matrilin-3 and -1 A-domains onto immobilized type IX collagen (left panel). The right panel shows the D-f plot using QCM-D raw data at the third overtone (n = 3).
Mentions: The M1A2 domain showed the strongest affinity with a KD of ∼50 nm compared with M3A (∼91 nm) and M1A1 (∼104 nm) (Fig. 5A). This trend was in agreement with the calculated KD as determined by SPR analyses, which were 2.13 nm for M1A2, 6.2 nm for M3A, and 8.8 nm for M1A1 (Fig. 4B, top panel, and table). The difference in binding between M1A2 and M3A was also statistically significant (p < 0.001). However, no difference was detected between wild type M1A2 and the unglycosylated form (probably due to the fact that wild type M1A2 contains a high proportion of unglycosylated protein resulting in a masking effect). An interesting finding was that the unglycosylated form of M1A1 bound more strongly to type II collagen than the wild type molecule. This suggests that the flexible N-glycan chains might partially hide the binding site for type II collagen, thus narrowing the location of the binding site for this molecule to the opposite face of the MIDAS motif (Fig. 2D).

Bottom Line: Initial characterization revealed that N-glycosylation did not affect secretion of these two proteins, nor did it alter their folding characteristics.However, removal of the glycosylation decreased their thermal stability.We then compared the effect of different cations on binding between both M1A domains and type II and IX collagens and showed that Zn(2+) also supports their interactions.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester M13 9PT, United Kingdom.

ABSTRACT
Matrilin-1 is expressed predominantly in cartilage and co-localizes with matrilin-3 with which it can form hetero-oligomers. We recently described novel structural and functional features of the matrilin-3 A-domain (M3A) and demonstrated that it bound with high affinity to type II and IX collagens. Interactions preferentially occurred in the presence of Zn(2+) suggesting that matrilin-3 has acquired a requirement for specific metal ions for activation and/or molecular associations. To understand the interdependence of matrilin-1/-3 hetero-oligomers in extracellular matrix (ECM) interactions, we have extended these studies to include the two matrilin-1 A-domains (i.e. M1A1 and M1A2 respectively). In this study we have identified new characteristics of the matrilin-1 A-domains by describing their glycosylation state and the effect of N-glycan chains on their structure, thermal stability, and protein-protein interactions. Initial characterization revealed that N-glycosylation did not affect secretion of these two proteins, nor did it alter their folding characteristics. However, removal of the glycosylation decreased their thermal stability. We then compared the effect of different cations on binding between both M1A domains and type II and IX collagens and showed that Zn(2+) also supports their interactions. Finally, we have demonstrated that both M1A1 domains and biglycan are essential for the association of the type II·VI collagen complex. We predict that a potential role of the matrilin-1/-3 hetero-oligomer might be to increase multivalency, and therefore the ability to connect various ECM components. Differing affinities could act to regulate the integrated network, thus coordinating the organization of the macromolecular structures in the cartilage ECM.

Show MeSH