To investigate the presence of cotranslationally formed disulfide bonds, the translation reaction was carried out in the presence of GSSG. GSSG oxidizes existing thiol groups and therefore facilitates the formation of disulfide bonds. It was required in the reaction mixture to compensate for the presence of the reducing agent dithiothreitol in the commercially prepared reticulocyte lysates. Varying concentrations of GSSG were added to the reticulocyte lysates before initiation of translation to obtain a lysate that was competent in forming disulfide bonds in the newly synthesized protein. A change in the thiol/disulfide redox status of the translation products has been shown to influence the migration pattern of many proteins (
- Goldenberg D.P.
- Creighton T.E.
). However, this could not be assessed electrophoretically for brush border LPHm (Fig. 1
), LPH isolated from biopsy samples (see Fig. 4
), or LPH synthesized in a cell-free system (data not shown). We therefore sought to determine at the immunochemical level whether GSSG induced conformational alterations in pro-LPH reminiscent of the formation of native disulfide bonds. Here, we used two antibodies directed against LPH: a conformation-specific monoclonal anti-LPH antibody (anti-LPH mAb) (
- Hauri H.-P.
- Sterchi E.E.
- Bienz D.
- Fransen J.A.M.
- Marxer A.
) and a polyclonal antibody directed against the denatured form of LPH (anti-LPH pAb). In the absence of GSSG, immunoprecipitation of the translation products with anti-LPH mAb did not reveal molecular species corresponding to pro-LPH (Fig. 3A
, lane 1
), although the translation products contained pro-LPH molecules (Fig. 3B
, lane 1
). In fact, anti-LPH pAb clearly recognized pro-LPH species that were translated in the presence or absence of GSSG (Fig. 3B
). On the other hand, in the presence of GSSG, a dose-dependent reactivity of pro-LPH with anti-LPH mAb was manifested. The intensity of pro-LPH (Mr
= 215,000) increased when the concentration of GSSG was raised from 1 mM (Fig. 3A
, lane 2
) to 2 mM (lane 3
), but decreased at higher concentrations (up to 4 mM) (lanes 4
). Comparison of the labeling intensities by scanning densitometry of immunoprecipitated pro-LPH (Fig. 3A
) and total pro-LPH in the translation products (data not shown) revealed that the decrease in the intensity of immunoprecipitated pro-LPH was due to inhibition of protein synthesis by GSSG and not due to unfolding of pro-LPH. In fact, Fig. 3D
demonstrates that the ratio of immunoprecipitated pro-LPH at a certain GSSG concentration versus
total synthesized pro-LPH at the same GSSG concentration increased when the concentration of GSSG was raised from 1 to 2 mM, but reached a plateau at higher GSSG concentrations. This result therefore indicates that anti-LPH mAb immunoprecipitates similar proportions of pro-LPH from total pro-LPH synthesized in the presence of 2, 3, and 4 mM GSSG. This strongly suggests that the epitope recognized by the mAb in the pro-LPH molecule does not undergo significant conformational alterations at GSSG concentrations between 2 and 4 mM.
On the other hand, it is possible that GSSG may influence the binding of the mAb to the pro-LPH species at increasing concentrations of GSSG, thus leading to reduced amounts of immunoprecipitated pro-LPH at concentrations higher than 2 mM. To investigate this issue, we labeled biopsy samples biosynthetically for 2 h, after which time only the 215-kDa mannose-rich pro-LPH species becomes labeled (
- Naim H.Y.
- Sterchi E.E.
- Lentze M.J.
). The detergent extracts of the biopsy samples were then treated with various concentrations of GSSG and immunoprecipitated with anti-LPH mAb. As shown in Fig. 3C
, essentially similar amounts of labeled pro-LPH were immunoprecipitated at increasing concentrations of GSSG. Therefore, this result demonstrates that GSSG per se
does not affect antibody-antigen binding. Consequently, these and the scanning data are consistent with the view that the decrease in the labeling intensity of immunoprecipitated pro-LPH (Fig. 3A
) is due to reduced amounts of translated pro-LPH at high GSSG concentrations. Taken together, the reactivity of the conformation-specific antibody with pro-LPH generated in the presence of GSSG, but not in its absence, strongly suggests that the oxidant facilitates the folding of pro-LPH by formation of disulfide bonds, whereby pro-LPH ultimately assumes a conformation similar to that of native intestinal pro-LPH.