Concurrent ossification of the ligamentum flavum (OLF) in the cervical, thoracic, and lumbar spine is a rare occurrence often associated with rheumatologic abnormalities.
The 200 210 nm range is more positive at pH 4.6 than the others, suggestive of non-native features of OLF in this buffer environment, but otherwise there are only slight differences in the intensity of the two major minima at ∼217 and ∼230 nm (Figure 2A).
The addition of dermatan sulfate did not change the Tm value of myoc-OLF in the full range tested.
Selected DSF measurements were repeated with cleaved myoc-OLF in acetate, MES, phosphate, Tris, bicine, and glycine buffers.
Thus, we sought to evaluate the stability of myoc-OLF in the presence of individual GAGs found in the TEM [23] at physiologically relevant concentrations [24] (Table 2).
To cross-validate the results obtained using the MBP-OLF fusion protein, we subsequently tested cleaved myoc-OLF in a subset of stabilizing and destabilizing buffers (Table S1).
Our experiments reveal a modest increase in stability for myoc-OLF in the presence of heparan sulfate, chondroitin sulfate, and hyaluronic acid.
The optimal reaction condition consisted of a 1∶200 dilution of subtilisin A (Sigma Aldrich, 1 mg/ml) in 0.2 mg/ml myoc-OLF in 10 mM Hepes pH 7.5 or 1∶200 dilution of papain (Sigma, 1 mg/ml) in 0.2 mg/ml myoc-OLF in 10 mM MES pH 6.2.
The reaction mixture consisted of 1 µM myoc-OLF or MBP-OLF in 10 mM sodium phosphate dibasic/potassium phosphate monobasic, 200 mM NaCl pH 7.2 (Buffer A) and 5X Sypro Orange (Invitrogen).
Indeed, a modest (ΔTm = 4.5°C) stabilizing effect was observed for myoc-OLF in the presence of a relatively low concentration (0.75 mg/ml) of heparan sulfate, whereas a similar increase in stability for chondroitin sulfate and hyaluronic acid were observed only at higher concentrations of 10 mg/ml, at the outer limit of the physiological range, suggesting a weaker effect.
In addition, although heparan sulfate appears to exert a modest stabilizing effect on myoc-OLF in the absence of other analytes, it seems plausible that cations might enhance the favorable specific interactions between the negatively charged GAGs and the negatively charged OLF domain under physiological conditions.
