a rapid pro ximal magnit d HDL show in nHDL. L DATA. A on the L t Function ... d protein m of SAA. HD lar ratios (re rotein ratio o min) and pos ns at various.
SUPPLE EMENTAL L DATA
Paradoxical Effects s of Serum m Amyloid A on the Lipoprotein n Oxidation n Sugges st a New Antioxidant A t Function for SAA
Shobini Jayaraman,, Christian H Haupt, and O Olga Gursky
Figurre S1. Lipid peroxidation kinetics by y copper in native HDL L, nHDL (bla ack), and in SAAenriched HDL, SAA A·HDL(total) (gray). Lipo oproteins we ere prepare ed as descrribed in Figu ure 2 T charactteristic lag followed by a rapid pro pagation ph hase (with th he maximal rate, legend. The Vmax) and d a saturatio on phase (w with the max ximal magnittude, ODmax) are indicatted. An exte ended time scale (0-300 min) for the SAA-enriched S d HDL show ws that the m maximal magnitude of O OD at saturation, ODmax, is much smaller than that in nHDL.
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S Lipid and protein modification m ns in HDL o oxidized to v various stages show d doseFigure S2. depende ent effects of o SAA. HD DL that conta ained variou s amounts o of SAA, from m 0.5:1 (blacck) to 2:1 SAA A:apoA-I mollar ratios (re ed), were oxidized by C Cu2+ to variious stages as described in Figure 2A A. For the prrotein ratio of o 0:1 or 0.5:1, these sta ages corresp pond to the pre-transitio on (15 min), tran nsition (45 min) m and pos st-transitiona al regions (10 00 min) in th he OD234 currve (Fig. 2A)). (A) Lipid modification ns at various s oxidation stages s were monitored b by UV/vis ab bsorption sp pectra at 25 °C C reporting on the ca arotenoid co onsumption.. The finge erprint abso orption peakks of caroteno oids at 485, 458 4 and 434 4 nm progre essively disa appear upon oxidation a at 0.5:1 but n not at 2:1 ratio. Consequen ntly, at 2:1 ra atio SAA effe ectively delayys carotenoid consumpttion in HDL ccore. (B) Prottein modific cations at various v oxidation stage s were monitored by the intrinsicc Trp fluoresce ence of HDL L. The spectrra were reco orded as desscribed in M Methods. At 0 0.5:1 SAA:apoA-I ratio, pro ogressive de ecrease in the t emission n intensity o of Trp togetther with the e red shift in the emission n maximum
indicate protein p modifications su uch as Tpr oxidation m mediated byy the
products of lipid ox xidation. At 2:1 SAA:apoA-I ratio, these spe ectral chang ges were grreatly S delays oxidative m odifications to HDL protteins. retarded.. Therefore, at 2:1 ratio SAA
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S Characte erization off the protein released from oxidizzed SAA·HD DL. SEC fra action Figure S3. of the prrotein releas sed from mildly SAA·HD DL at 2:1 SA AA:apoA-I m molar ratio ((marked “fre ee” in Figure 3)) were collec cted and ana alyzed. (A) SDS PAGE of th he protein re eleased from m HDL show ws SAA and apoA-I band ds. Quantificcation ands from three indepe endent gels was carried d out as pre eviously desscribed (30)); the of the ba results sh howed appro oximately 40 0% apoA-I and 60% SAA A by weight.. (B) MALDI-TOF ana alysis of the protein rele eased from ox·SAA·HD DL. The specctrum showss two peaks wiith the mass s of 11,656 Da D and 28,1 135 Da corre esponding to o Met-oxidizzed forms off SAA and apo oA-I, respec ctively. The spectra of o non-oxidizzed SAA a and apoA-I are shown n for comparis son (bottom panel).
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Figurre S4. Statis stical analy ysis of the effects e of S SAA on the oxidation o of HDL and LDL. HDL incu ubation mixttures, terme ed HDL(total), contained d various am mounts of S SAA as show wn in Figure 4A A, from 0:1 to 4:1 SAA::apoA-I mol ratios. LDL were eitherr alone or in n the presen nce of SAA or apoA-I a at 1:1 SAA:apoB B or apoA-I::apoB weigh ht ratios (Fig g. 4B). Thesse samples were oxidized by Cu2+ as described d in n Figure 4 an nd the OD2344 data were recorded (Fig. 4A). The e HDL data sho owed a complete sigmo oidal transitio on at 0:1, 0 0.5:1 or 1:1 SAA:apoA-II molar ratio os (at higher SA AA;apoA-I ratios r the tra ansition was s incomplete e and hence e, was not a analyzed furrther). The expe eriments we ere repeated d 3 times. The T transition n midpoint, t½ (in min) a and the maximal rate, Vmaax (in OD units per min) were determ mined from the sigmoid dal fitting of the OD234(t)) data and its first f derivative. The res sults were subjected s to o a statisticcal analysis as describe ed in Methods. LDL oxidation data we ere analyzed d similarly. T The results of the statisstical analyssis for s of t½ and V max for HDL and LDL arre presented d as a HDL (A) and LDL (B) are shown. The values pendent mea asurements, with the er ror bars sho owing the sttandard deviation mean of three indep ean. P-value es are indica ated: * (P
