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Background: The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation., Methods: Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions., Results: Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of 'gelatinolytic' activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity., Conclusions: Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic., General Significance: The study suggests that a bi-directional cause-effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.

Refolding of the heavy chain of the Class I HLA molecule, HLA-B27, in the absence of beta(2)m, yields soluble high molecular weight (HMW) oligomers reminiscent of the oligomeric forms of beta(2)m-free heavy chains (FHCs) of class I HLA antigens observed on cell surfaces. Here we examine the structural characteristics of HMW B27 in respect of features potentially relevant to autoimmunity, such as: (a) retention of native-like structure, since this could facilitate non-canonical interactions with T-cell receptors even in the absence of bound beta(2)m and peptide, or (b) presence of non-native structure, since this could yield novel (non-self) antigenic conformational epitopes that could elicit immune attack. We report that HMW B27 is characterized by high secondary structural content, structural stability, stability to proteolysis by trypsin, and structural features that are both partly native-like, and partly non-native-like, as assessed through the binding of conformationally-distinguishing and cross-reacting scFv antibodies specifically selected against HMW B27. We also present cell ELISA data with conformation-specific scFv antibodies that distinguish between lymphocytes from individuals who are healthy and B27 positive, and those who are B27 positive but suffering from ankylosing spondylitis.