Shotgun Protein Profile of Human Adipose Tissue and Its Changes in Relation to Systemic Amyloidoses

In systemic amyloidosis, accumulation of misfolded proteins as extracellular amyloid fibrils in tissues causes severe organ dysfunction, but the molecular events of tissue damage related to amyloid deposition are still largely unknown. Through the use of the MudPIT proteomic approach, comprehensive protein profiles of human amyloid-affected adipose tissue from patients and its control (non-amyloid-affected) counterpart were acquired. Label-free comparison between patients and controls made it possible to highlight differences related to the presence of amyloid, by describing up- and down-represented proteins, connected into interacting networks. In particular, extracellular matrix (ECM), protein folding, lipid metabolism, and mitochondrial functions were among the most affected structural/functional pathways. The reported results, obtained with no a priori hypotheses, represent a significant step forward in the clarification of the molecular mechanisms involved in amyloidoses at tissue level and are the premise for understanding protein misfolding diseases

Intact mass determination of purified rLC by MALDI-TOF mass spectrometry.

<p>Panel A shows the peaks corresponding to the single charged ions. Panel B shows the calculated (including the presence of an N-terminal methionine) and the observed intact masses of each LC. The error (ppm) from the calculated mass is reported. The results are consistent with the predicted molecular masses.</p

Size exclusion chromatography of rLC.

<p>rAL-1 (dotted), rAL-2 (short dashes), rAL-3 (dash-dot-dot) compared with AL-2 BJ protein (solid). Conalbumin (75.0 kDa), ovalbumin (44.0 kDa), carbonic anhydrase (29.0 kDa), ribonuclease A (13.5 kDa), aprotinin (6.5 kDa) were used as molecular mass standards. Virtually identical elution profiles were observed.</p

SDS–PAGE analysis of the purified rLC.

<p>Coomassie Brilliant Blue staining (A) and western blotting (B) analysis of purified rLC compared to AL-2 Bence Jones protein (AL-2 BJ), purified from urine. Protein samples (5 µg loaded) were resolved on 12% SDS-PAGE run under non reducing and reducing conditions. Western blot analysis was performed using a rabbit anti-human λ LC antiserum as primary antibody (Dako Cytomation, Glostrup, Denmark), revealed by a goat anti-rabbit conjugated with horseradish peroxidase (Dako Cytomation), probed with diaminobenzidine (DAB) substrate. Molecular mass markers are shown in the left lane. The proteins presented the typical dimeric-monomeric species of free LC. Different degrees of protein denaturation are responsible of the minor differences in electrophoretic migration of monoclonal free LC (A), a common finding in SDS PAGE running under denaturing conditions of these proteins.</p

rLC <i>in vitro</i> fibrillogenesis. Congo-red binding profiles and electron microscopy.

<p>Panel A reports Congo-red binding patterns during fibrillogenesis. rAL-2 and AL-2 BJ revealed similar binding profiles. Panel B shows electron microscopy of typical amyloid fibrils generated <i>in vitro</i> by the rLC (fibrils from rAL-2 are shown). Magnification is 60000 x.</p

Schematic representation of the expression strategy.

<p>Each patient’s full-length monoclonal LC (V_L +C_L) obtained by RT-PCR was re-amplified using primers designed according to the pASK-IBA cloning strategy. Start Met codon in 5′ primer and the two stop codons in 3′ primer were introduced and shown in bold, <i>Bsm</i>BI recognition sites are indicated in lower case in both primers. After <i>Bsm</i>BI digestion, PCR product was ligated into <i>Bsa</i>I/<i>Bsa</i>I sites of pASK-IBA33plus vector.</p

Circular dichroism analyses.

<p>Far-ultraviolet CD spectra (panel A, pH 7.4; panel C, pH 5.0) of purified rAL-1 (short dashes), rAL-2 (dotted), rAL-3 (solid) compared with the spectrum of patient AL-2 BJ protein (dash-dot-dot). Each CD spectrum represents the average of ten scans. Data are shown as mean residue ellipticity (MRE) as a function of wavelength. Panels B and D report the secondary structure content of the analyzed proteins (at pH 7.4 and 5.0, respectively), calculated with K2D, CDSSTR and CONTIN software applications. Data are expressed as mean ± sd. Spectra were virtually identical for rAL-2 and the corresponding natural BJ (panels A and C), overlapping for the other two rLC (94% amino acid identity), and display the minimum at around 218 nm, characteristic of β-sheets. Proteins showed very similar percentage of secondary structures.</p