Severe Isotype-Matched Immunosuppression (IMI) as a Potential Risk Factor for Progression of MGUS Patients.

BACKGROUND: Monoclonal gammopathy of undetermined significance (MGUS) precedes multiple myeloma in virtually every case. However, only a small percentage will progress and at very different rates. In addition, recent data have suggested that MGUS is associated with other comorbidities including infections, suggesting impaired immune function in some MGUS patients. Therefore, we aimed at assessing the value of isotype-matched immunosuppression (IMI; e.g., suppression of an IgAκ in an IgAλ patient), a type of immunosuppression more specific than classical immunoparesis (IP; e.g., IgG and/or IgM suppression in an IgA patient), as a prognostic marker for MGUS progression. METHODS: The Hevylite assay was used to assess IMI and immunoglobulin ratios in 307 serum samples from a cohort of 248 MGUS patients. Follow-up clinical records were available for 154 individuals. RESULTS: A greater incidence of IMI (51%) over classical IP (37%) was observed, although both show a progressive increase with higher risk groups. Survival analysis of 154 patients showed that severe IMI (>50% suppression) differentiates 2 groups with significantly different time to progression (P = 0.024) while severe IP does not (P = 0.48). Also, a combination of severe IMI and involved monoclonal immunoglobulin >1.5g/dL by Hevylite (both variables found to be independent prognostic markers in multivariate analysis) identified a group of patients with a median time to progression 6-fold shorter than the remaining group (P < 0.0001). CONCLUSIONS: These findings indicate a possible role for IMI in the malignant transformation of MGUS patients and a potential utility as a new risk factor.

Mannosylglycerate stabilizes staphylococcal nuclease with restriction of slow ß-sheet motions.

Mannosylglycerate is a compatible solute typical of thermophilic marine microorganisms that has a remarkable ability to protect proteins from thermal denaturation. This ionic solute appears to be a universal stabilizing agent, but the extent of protection depends on the specific protein examined. To understand how mannosylglycerate confers protection, we have been studying its influence on the internal motions of a hyperstable staphylococcal nuclease (SNase). Previously, we found a correlation between the magnitude of protein stabilization and the restriction of fast backbone motions. We now report the effect of mannosylglycerate on the fast motions of side-chains and on the slower unfolding motions of the protein. Side-chain motions were assessed by (13)CH(3) relaxation measurements and model-free analysis while slower unfolding motions were probed by H/D exchange measurements at increasing concentrations of urea. Side-chain motions were little affected by the presence of different concentrations of mannosylglycerate or even by the presence of urea (0.25M), and show no correlation with changes in the thermodynamic stability of SNase. Native hydrogen exchange experiments showed that, contrary to reports on other stabilizing solutes, mannosylglycerate restricts local motions in addition to the global motions of the protein. The protein unfolding/folding pathway remained undisturbed in the presence of mannosylglycerate but the solute showed a specific effect on the local motions of ß-sheet residues. This work reinforces the link between solute-induced stabilization and restriction of protein motions at different timescales, and shows that the solute preferentially affects specific structural elements of SNase.

Relationship between protein stabilization and protein rigidification induced by mannosylglycerate.

Understanding protein stabilization by small organic compounds is a topic of great practical importance. The effect of mannosylglycerate, a charged compatible solute typical of thermophilic microorganisms, on a variant of staphylococcal nuclease was investigated using several NMR spectroscopy methods. No structural changes were apparent from the chemical shifts of amide protons. Measurements of (15)N relaxation and model-free analysis, water-amide saturation transfer (phase-modulated CLEAN chemical exchange), and hydrogen/deuterium exchange rates provided a detailed picture of the effects of mannosylglycerate on the backbone dynamics and time-averaged structure of this protein. The widest movements of the protein backbone were significantly constrained in the presence of mannosylglycerate, as indicated by the average 5-fold decrease of the hydrogen/deuterium exchange rates, but the effect on the millisecond timescale was small. At high frequencies, internal motions of staphylococcal nuclease were progressively restricted with increasing concentrations of mannosylglycerate or reduced temperature, while the opposite effect was observed with urea (a destabilizing solute). The order parameters showed a strong correlation with the changes in the T(m) values induced by different solutes, determined by differential scanning calorimetry. These data show that mannosylglycerate caused a generalised reduction of backbone motions and demonstrate a correlation between protein stabilization and protein rigidification.

Structural determinants of protein stabilization by solutes. The important of the hairpin loop in rubredoxins.

Despite their high sequence homology, rubredoxins from Desulfovibrio gigas and D. desulfuricans are stabilized to very different extents by compatible solutes such as diglycerol phosphate, the major osmolyte in the hyperthermophilic archaeon Archaeoglobus fulgidus[Lamosa P, Burke A, Peist R, Huber R, Liu M Y, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C and Santos H (2000) Appl Environ Microbiol66, 1974-1979]. The principal structural difference between these two proteins is the absence of the hairpin loop in the rubredoxin from D. desulfuricans. Therefore, mutants of D. gigas rubredoxin bearing deletions in the loop region were constructed to investigate the importance of this structural feature on protein intrinsic stability, as well as on its capacity to undergo stabilization by compatible solutes. The three-dimensional structure of the mutant bearing the largest deletion, Delta17/29, was determined by 1H-NMR, demonstrating that, despite the drastic deletion, the main structural features were preserved. The dependence of the NH chemical shifts on temperature and solute concentration (diglycerol phosphate or mannosylglycerate) provide evidence of subtle conformational changes induced by the solute. The kinetic stability (as assessed from the absorption decay at 494 nm) of six mutant rubredoxins was determined at 90 degrees C and the stabilizing effect exerted by both solutes was assessed. The extent of protection conferred by each solute was highly dependent on the specific mutant examined: while the half-life for iron release in the wild-type D. gigas rubredoxin increased threefold in the presence of 0.1 M diglycerol phosphate, mutant Delta23/29 was destabilized. This study provides evidence for solute-induced compaction of the protein structure and occurrence of weak, specific interactions with the protein surface. The relevance of these findings to our understanding of the molecular basis for protein stabilization is discussed.