Fusion of EFEMP1 to GLuc serves as a convenient and sensitive luminescence method to monitor EFEMP1 secretion
As an alternative to pulse chase experiments used previously to quantify EFEMP1 secretion (Marmorstein et al., 2002
), we fused EFEMP1 to GLuc to report on its extracellular versus intracellular localization. To test whether the EFEMP1-GLuc fusion proteins were secreted similar to what was reported for wild-type (WT) and R345W EFEMP1, HEK-293T cells were transiently transfected with EFEMP1-GLuc–encoding constructs and the secretion of WT EFEMP1-GLuc and R345W EFEMP1-GLuc was followed over time by assaying for GLuc activity in the media. Three hours after a media change (19 h after transfection), WT and R345W EFEMP1-GLuc were detectable in the media (). At 3 h, ~13 ± 3.2–fold more WT EFEMP1-GLuc was observed relative to R345W EFEMP1-GLuc (). The WT and R345W EFEMP1-GLuc media concentrations continued to increase linearly over the time course of the experiment, with WT EFEMP1-GLuc levels peaking at ~16 ± 0.36–fold higher than those of R345W EFEMP1-GLuc 24 h after the media change ().
To assess the sensitivity of the GLuc assay, we performed Western blotting on conditioned media samples. Whereas the luciferase assay detected WT EFEMP1-GLuc as early as 3 h after the media change, Western blotting was only able to detect the fusion protein 9 h after the media change, corresponding to a luciferase assay readout of ~100,000 ALU, a signal ~200 times greater than our designated minimum detectible GLuc signal of 500 ALU (compare to ). Secreted R345W EFEMP1-GLuc accumulated to a much lower extent in the media than WT EFEMP1-GLuc by Western blot analysis (), consistent with quantification using the GLuc assay (). Of note, the levels of R345W EFEMP1-GLuc barely reached the limit of detection using Western blotting ().
To ensure that the folding and secretion differences we observed between WT and R345W EFEMP1-GLuc were not due simply to differences in transfection efficiency or transcript levels, we performed quantitative real-time PCR. No significant differences in transcript levels of EFEMP1 or GLuc were observed in HEK-293T cells transfected with either WT or R345W EFEMP1-GLuc (Supplemental Figure S1, A and B). In general, EFEMP1 transcript levels were found to be 100–300 times higher in cells transfected with EFEMP1-GLuc plasmids than in untransfected controls, indicating significant EFEMP1 overexpression (Supplemental Figure S1A).
Aromatic amino acid substitutions at position 345 result in inefficient EFEMP1 secretion
To gain insight into the molecular basis for the poor secretion of R345W EFEMP1, side chain hydrophobicity, size, charge, and side chain influence on backbone conformation were varied at the R345 position by mutagenesis. We generated 10 position 345 variants of EFEMP1-GLuc (in addition to R345W) and monitored the secretion efficiency of these variants relative to WT EFEMP1-GLuc 24 h after a media change (72 h after transfection; ). Mutation from the native arginine residue, which is positively charged at physiological pH, to an uncharged residue bearing a relatively small side chain (R345A) or to an uncharged residue bearing no side chain and exhibiting maximal local backbone conformational flexibility (R345G) had no detrimental effect on secretion efficiency relative to WT EFEMP1-GLuc (86 ± 28% and 92 ± 22%, respectively; ). Altering the Arg side chain to a similarly sized polar and conformationally flexible side chain, including uncharged glutamine (R345Q, 89 ± 17%; ) or charged lysine (R345K, 91 ± 25%) did not reduce folding and secretion efficiency notably either. Mutation to a similarly sized, hydrophobic uncharged side chain (leucine, R345L, 70 ± 15%; ) slightly reduced secretion compared with WT EFEMP1-GLuc. Surprisingly, reversal of the positive charge on the side chain of arginine to the negative charge on the side chain of glutamic acid (R345E) did not lower EFEMP1 secretion substantially either (78 ± 21%; ). In contrast, mutating R345 to a large aromatic side chain, including tyrosine (R345Y, 15 ± 3.4%; ) and phenylalanine (R345F, 20 ± 5.0%) dramatically reduced secretion efficiency, analogous to what was observed with the R345W pathological EFEMP1 mutant (10 ± 4.7%; ). Mutation of R345 to the smaller heteroaromatic imidazole side chain of histidine (R345H) also reduced EFEMP1 secretion (69 ± 12%; ), albeit to a lesser extent than the larger aromatic side chains. Mutation of R345 to an uncharged cyclic proline residue (R345P) was almost as devastating to EFEMP1 secretion as mutation to phenylalanine (22.4 ± 6.3%; ), probably due to the restricted backbone conformation of proline (ϕ = −65°; Williamson, 1994
FIGURE 2: GLuc luminescence assay of secreted WT EFEMP1-GLuc and R345 EFEMP1-GLuc mutants transfected into HEK-293T cells. (A) Aromatic amino acid substitutions at R345 cause secretion defects. Aliquots of 45 μl of conditioned media from transfected HEK-293T (more ...)
To eliminate a possible alternative explanation for the secretion differences observed among the variants of EFEMP1-GLuc studied, we assessed the cell toxicity caused by these mutants, as cytotoxicity would lead to an apparent decrease of secretion of EFEMP1 into the media. Cellular viability was assessed 72 h after transfection by employing the 7-hydroxy-3H-phenoxazin-3-one 10-oxide (resazurin) assay, which reports on mitochondrial redox potential. On the basis of this often-used assay for cytotoxicity (Usui et al., 2009
), none of the R345 EFEMP1-GLuc mutants generated were toxic to HEK-293T cells ().
We also controlled for the possibility that transfection efficiency differences could explain lower apparent secreted EFEMP1 mutant levels. To explore this possibility, another secreted luciferase (Cypridina luciferase [CLuc]) was cotransfected with the EFEMP1-GLuc constructs (Supplemental Figure S2). The secretion trends described earlier without CLuc normalization () persisted with CLuc normalization (Supplemental Figure S2), demonstrating that none of the trivial explanations for the secretion differences observed appear to be valid. The CLuc cosecretion data also demonstrate that mutant EFEMP1-GLuc does not nonspecifically reduce secretion capacity from the ER under these transient transfection conditions.
Mutations leading to inefficient secretion of EFEMP1 are underrepresented at the bn + 1 position of other Ca2+-binding EGF domains in the fibulin family
The pathogenic EFEMP1 mutation position is located immediately adjacent to cysteine residue 344 (C344), which is required to form disulfide bond 2 of domain 6 (dsb2; ). Disulfide bond 2 is predicted to be critical for the folding and function of the Ca2+
-binding EGF domain (Chang et al., 1995
). We hypothesized that compromised disulfide bond formation could play a part in reducing R345W EFEMP1 secretion efficiency. Comparative analysis of the amino acid residues present at the bn
+ 1 location in 40 nonmutated Ca2+
-binding EGF domains from six different fibulin proteins indicates that none contain tryptophan, tyrosine, or phenylalanine and only one contains proline (Supplemental Tables SI and SII). Thus, of the four residues at position 345 that cause defects in EFEMP1 secretion (Trp, Phe, Tyr, and Pro), only proline is found in one of the 40 domains analyzed (domain 4 in fibulin-1, Supplemental Tables SI and SII). If the amino acid type at position 345 were determined statistically, then these four residues (Trp, Phe, Tyr, Pro) would be expected to occupy eight of the 40 possible domains, yielding an occupancy rate of 20%, a number far greater than the observed occupancy of 2.5% at this position (Supplemental Table SII). Mutations at the bn
+ 1 position that modestly affect secretion (R345H, R345L, and R345E) are present in five domains (12.5%), close to the predicted 15% occupancy. Of the 34 remaining domains, 25 are preferentially occupied at the bn
+ 1 position by glutamine (nine domains), arginine (seven domains), or serine (seven domains), accounting for 62.5% of the residues at this position. Our data indicate that at least two of these three prominent amino acids are well tolerated at position 345 and have relatively similar secretion efficiencies (; serine not evaluated). Although the sequence analysis of the evolutionary record and experimental sequence-dependent secretion data correlate well, an exception is lysine, which is not found in the bn
+ 1 position in any of the 40 domains studied (Supplemental Table SI and SII), yet its secretion efficiency is indistinguishable from that of WT ().
Inefficient secretion by EFEMP1 mutants is improved by lowered cell growth temperatures
The misfolding/aggregation and/or increased steady-state retention of several disease-associated proteins at 37°C within cells can be rescued by reducing the cellular growth temperature from 37 to 27–33°C (Kulka et al., 1988
; Michalovitz et al., 1990
; Denning et al., 1992
; Wang et al., 2008
). To determine whether the poor secretion of the R345W mutation could be improved by a shift to lower temperatures, we reduced the growth temperature of WT- or mutant-expressing HEK-293T cells to 30°C for up to 24 h. Within 3 h of the temperature shift, significantly more R345W (relative to WT) was secreted to the media, increasing from 11 to 24% of WT levels (). Secretion was enhanced over the span of 24 h, peaking at ~53% of WT secretion at 24 h, whereas R345W secretion at 37°C remained at ~10% of WT levels under identical conditions ().
FIGURE 3: Secretion of EFEMP1 mutants is temperature sensitive. (A) HEK-293T cells were transfected for 48 h at 37°C, followed by a media change and temperature shift to 30°C for up to 24 h. Aliquots were taken at the indicated intervals after the (more ...)
We also examined whether the cellular secretion of the additional R345 point mutants was temperature sensitive. A lower (permissive) temperature of 30°C significantly enhanced the secretion of the R345Y, R345F, R345P, R345L, and R345E variants, raising the relative secretion percentage from a range of 10–82% at 37°C to 47–104% at 30°C 24 h after temperature shift (). Of interest, R345W was still secreted most poorly, followed by R345Y and R345F (). A comparison of the total amount of EFEMP1 secreted at 24 h based on measured luminescence values (not relative to WT) is presented in Supplemental Figure S3, revealing a strictly analogous picture.
Identically treated samples were subjected to Western blot analysis () to verify the validity of the GLuc assay results (). At 37°C, the inefficiently secreted and presumably misfolded EFEMP1 mutants, R345W, R345Y, R345F, and R345P were barely detectable (). In agreement with the GLuc assay, levels of these fusion proteins in conditioned media were significantly enhanced by growth at the permissive temperature (). Quantification of the integrated band intensities was performed, revealing that the GLuc luminescence and Western blot band intensities were in agreement (Supplemental Figure S4A). Furthermore, GLuc luminescence correlated linearly with EFEMP1 protein concentration, as determined by LI-COR quantification (Supplemental Figure S4B).
Inefficiently secreted variants accumulate intracellularly
To account for the remainder of the R345 aromatic- and R345 proline-containing EFEMP1-GLuc mutants that was not secreted, we also assessed their intracellular abundance. The four mutants that exhibited the largest secretion deficiencies accumulated intracellularly up to two times the levels of WT under identical steady-state conditions at 37°C, whereas intracellular levels of the remaining mutants hovered around WT levels (). Thus, consistent with observations made by other groups using complementary approaches (Marmorstein et al., 2002
), it appears that the poorly secreted mutants misfold/aggregate and thus exhibit higher steady-state intracellular levels while awaiting degradation, most likely by ER-associated degradation (Werner et al., 1996
; McCracken et al., 1998
). Of interest, the same intracellular steady-state accumulation trends are observed in cells grown at 30°C; however, the absolute levels of intracellular EFEMP1-GLuc are much lower, most likely due to enhanced secretion (removal of the intracellular protein pool) in combination with translational attenuation (). Intracellular mutant EFEMP1-GLuc retention relative to WT EFEMP1-GLuc at 30°C is depicted in Supplemental Figure S5, demonstrating a similarity in trends when compared with .
Translational attenuation slightly increases mutant EFEMP1-GLuc secretion
One proteostasis network adaptation that can occur at lower cellular growth temperatures is translational attenuation (Fujita, 1999
). To address whether translational attenuation might contribute to the enhanced secretion of EFEMP1 mutants, transfected cells were treated with cycloheximide (CHX), which nonspecifically prevents translational elongation (Siegel and Sisler, 1963
). Addition of a low concentration of CHX (1 μM) for 24 h did not result in significant cell death, acting as a means to more gently prevent protein translation when compared with typical CHX treatments (up to 50 μM for a few hours). CHX did not significantly affect the secretion of WT EFEMP1-GLuc at 37°C (). However, intracellular levels of WT decreased after CHX treatment, indicating a reduction in protein translation and a removal of protein from the intracellular pool (). These results suggest that the majority of secreted EFEMP1-GLuc over the period of the 24-h time course originates from the intracellular pool of EFEMP1 already synthesized (not newly synthesized). Of interest, treatment of cells expressing WT EFEMP1-GLuc with CHX in combination with temperature reduction caused a significant reduction in secreted WT protein (to 65% of untreated WT levels; ). Addition of CHX to cells expressing R345W EFEMP1-GLuc cultured at 37°C significantly enhanced R345W secretion by greater than twofold over untreated R345W-expressing cells at the same temperature (13 ± 1.7% without CHX vs. 28 ± 11% with CHX; ). However, the extent of CHX-mediated rescue at 37°C was much lower than permissive temperature-mediated rescue (). Combining CHX and permissive temperature did not provide a synergistic or additive effect (), likely because temperature reduction already results in translational attenuation.
FIGURE 4: Translational attenuation with CHX subtly increases mutant EFEMP1 secretion. (A) HEK-293T cells were transfected with either WT or R345W EFEMP1-GLuc for 48 h at 37°C, followed by a media change and addition of 1 μM CHX and/or temperature (more ...)
To determine whether translational attenuation generally enhanced the secretion of poorly secreted mutants, we analyzed the effect of CHX on the secretion of the other R345 variants at 37°C. CHX treatment also significantly enhanced the secretion of other presumably misfolded/aggregated mutants, including R345Y, R345F, R345P, and R345L, yet had no effect on R345 mutants that normally secrete efficiently (R345A, R345Q, R345K, R345G; ).
Native disulfide bonds are required for proper EFEMP1 secretion
The formation of three disulfide bonds () is generally critical for the proper folding of EGF domains and other small, disulfide-rich proteins (Chang et al., 1995
; Bulaj and Goldenberg, 1999
). Specifically, we evaluated whether three native disulfide bonds were necessary for the folding and secretion of EFEMP1 (Sevier and Kaiser, 2002
). We systematically eliminated each disulfide bond in domain 6 using individual Cys-to-Ala mutants (C338A, C359A, and C377A) in the WT and R345W EFEMP1-GLuc backgrounds. All cysteine mutants in both sequence backgrounds showed a significant defect in secretion efficiency when compared with their respective controls (). In addition, intracellular steady-state levels of all cysteine variants were enhanced relative to WT, as expected (). To address the concern that the observed secretion deficiencies could be due to the generation of orphan cysteines that could form incorrect or nonnative disulfide bonds, we constructed a double-cysteine mutant (C344A/C359A) that is incapable of forming dsb2. This variant acted identically to the other, single-cysteine mutants (), demonstrating the necessity of dsb2 for proper EFEMP1 secretion.
FIGURE 5: Cysteine residues are required for proper EFEMP1 secretion and temperature sensitivity. (A) EFEMP1 secretion is negated by cysteine residue mutation. Aliquots of 45 μl of conditioned media from transfected HEK-293T cells were taken 72 h after (more ...)
Temperature reduction exhibited no positive effect on the secretion efficiency of any of the Cys-to-Ala EFEMP1 mutants (). These results demonstrate that a permissive temperature most likely improves the folding, secretion, and disulfide-bond formation fidelity of difficult-to-fold EFEMP1 mutants, but, of importance, a permissive temperature does not simply allow misfolded EFEMP1 or EFEMP1 missing a disulfide bond to exit the ER. This information, along with nonreducing Western blot analysis of secreted WT and R345W EFEMP1-GLuc (Supplemental Figure S6), suggests that the R345W and other EFEMP1-GLuc mutants that are secreted into the media most likely contain their native disulfide bonds and demonstrates the importance of proper disulfide bonding in domain 6 for proper folding and secretion.
2-(2-Nitrophenylsulfenyl)-3-methyl-3′-bromoindolenine cleavage of EFEMP1 reflects the presence or absence of dsb2 in domain 6
Within the WT EFEMP1-GLuc fusion protein, there are three tryptophan residues (see Supplemental Figure S7 for schematic). One is located near the amino terminus of EFEMP1 (W35). The second Trp is positioned near the carboxy terminus of the GLuc domain (corresponding to W652). The third Trp is W351 (, red arrow), which is located in domain 6 between the cysteine residues that form dsb2, the disulfide bond that we hypothesize is disrupted in the R345W mutant. We therefore reasoned that cleavage of mutant EFEMP1-GLuc fusion proteins with 2-(2′-nitrophenylsulfenyl)-3-methyl-3-bromoinolenine (BNPS-skatole), a reagent that cleaves the C-terminal peptide bond following tryptophan residues, might provide insight into the formation of disulfide bonds or lack thereof in domain 6 of mutant versus WT EFEMP1.
BNPS-skatole cleavage at W35 is predicted to have only a minimal effect on the molecular weight of the EFEMP1-GLuc fusion protein (a 2.2-kDa loss; see Supplemental Figure S7 for a schematic of the expected cleavage products with and without an intact dsb2). Cleavage after W652 in the GLuc portion of the fusion protein will not affect the molecular weight under nonreducing conditions, regardless of whether dsb2 is intact or not (Supplemental Figure S7), because W652 is bracketed by two cysteines (C561 and C657) that are predicted to form a disulfide bond in folded GLuc (Ferre and Clote, 2005
). Hence, the BNPS-skatole cleavage product of EFEMP1 with dsb2 intact is predicted to have a molecular weight of 70.1 kDa under nonreducing conditions (2.2 kDa smaller than the full-length 72.3 kDa; see Supplemental Figure S7). However, if dsb2 or an intermediate disulfide bracketing W351 is missing, the EFEMP1-GLuc fusion protein will dissociate into two halves upon BNPS-skatole cleavage, affording a C-terminal fragment with a molecular weight of 35.6 kDa (residues 352 onward) and an N-terminal fragment exhibiting a molecular weight of 34.5 kDa (containing residues 36–351 of EFEMP1). As predicted, substantially more of the C-terminal 35.6-kDa cleavage product formed in BNPS-skatole–treated R345W EFEMP1-GLuc lysates than in WT EFEMP1-GLuc HEK-293T lysates under identical conditions (). Production of the cleavage product was dependent on the presence of BNPS-skatole () and on disulfide bond formation, since WT EFEMP1 can form more of the cleavage products when its disulfide bonds are reduced (Supplemental Figure S8).
FIGURE 6: BNPS-skatole cleaves dsb2 in domain 6 of EFEMP1. (A) The domain 6 cleavage product is BNPS-skatole dependent. Lysates from transfected cells were treated overnight with either glacial acetic acid (−) or BNPS-skatole in glacial acetic acid (+) (more ...)
To identify which disulfide bond disruptions afford fragments on a nonreducing SDS–PAGE gel after BNPS-skatole cleavage, we examined lysates from cells transfected with the Cys-to-Ala EFEMP1 mutations. In the WT background, there was no significant increase in the BNPS-skatole cleavage products of the C338A and C377A variants when compared with WT (), suggesting that the lack of dsb1 or dsb3 does not significantly influence the levels of cleavage product. In contrast, the C359A mutant, which eliminates a cysteine required for dsb2 formation, produced significantly more cleavage product than the other Cys-to-Ala mutants, indicating that BNPS-skatole cleavage reports largely on the absence of dsb2 (), which is hypothesized to be incompletely formed in the R345W EFEMP1 mutant. It is intriguing that there is a portion of the C359A variant that is not cleaved after BNPS-skatole treatment. The remaining full-length C359A after BNPS-skatole cleavage is likely due to nonnative mixed disulfides, as increasing BNPS-skatole concentrations up to fourfold does not decrease full-length C359A band intensity (Supplemental Figure S9).
In the R345W background, the C338A mutation (eliminates dsb1) did not substantially alter the amount of BNPS-skatole cleavage product relative to R345W (). In agreement with the C359A mutation in the WT background eliminating dsb2, the R345W/C359A variant also generated significantly more cleavage product than did R345W (). These data suggest that some R345W EFEMP1-GLuc is capable of forming dsb2 (or bracketing intermediate disulfides), since without the C359A mutation substantially less R345W cleavage product is observed. Unexpectedly, the C377A mutation slightly lowered the amount of BNPS-skatole cleavage product relative to R345W (), possibly indicating stabilization of one or more disulfide intermediates that bracket both W345 and W351. As expected, the levels of BNPS-skatole cleavage product of the cysteine mutants were effectively unaltered by temperature reduction (Supplemental Figure S10).
Poorly secreted EFEMP1 mutants exhibit alterations in disulfide bond formation within domain 6 of EFEMP1
In cell lysates, all R345 mutant EFEMP1-GLuc constructs treated with BNPS-skatole exhibited detectible amounts of cleavage product (). The R345W mutation exhibited the most cleavage product () potentially due to the additional BNPS-skatole cleavage site, and/or less dsb2 formation, and/or fewer intermediate disulfide bonds surrounding the two tryptophans in domain 6 of R345W EFEMP1-GLuc (). Other poorly secreted mutants (R345Y, R345F, R345P) also exhibited a significantly higher level of the 35.6-kDa BNPS-skatole cleavage product when compared to R345G, a variant that secretes identically to WT (), also hinting at a decrease in dsb2 formation and/or a decrease in disulfide intermediates bracketing W351 in other poorly secreted EFEMP1 mutants. Growth at permissive temperatures had no significant effect on the amount of BNPS-skatole cleavage product relative to full-length EFEMP1 in cell lysates for both WT and R345W EFEMP1-GLuc (), suggesting that once EFEMP1-GLuc has achieved proper disulfide formation, it is efficiently removed from the EFEMP1 intracellular pool and secreted into the media.
FIGURE 7: Poorly secreted R345 mutants lack proper disulfide bond formation in domain 6. (A) Lysates from transfected HEK-293T cells were treated overnight with BNPS-skatole and separated on a nonreducing SDS–PAGE gel, followed by Western blotting as described (more ...)
To determine more definitively whether secreted R345W had an enhanced ability to form dsb2 (or a disulfide bond surrounding W345 and W351), we treated conditioned media from cells grown at reduced temperature with BNPS-skatole and monitored the 35.6-kDa cleavage product (Supplemental Figure S11A). Although intracellular R345W EFEMP1-GLuc generated ~600% more BNPS-cleavage product than did WT (), quantification of the fractional BNPS-skatole C-terminal cleavage product arising from secreted R345W EFEMP1-GLuc produced from cells grown at a permissive temperature suggests that R345W only has 86% more BNPS-skatole–sensitive cleavage product than WT (Supplemental Figure S11B), a substantial reduction in cleavage product when compared with intracellular R345W. These data, which may even overestimate the amount of cleavage in secreted R345W EFEMP1-GLuc relative to WT because of the miniscule amount of observed cleavage product (Supplemental Figure S11A), suggest that secreted R345W contains dsb2 or a disulfide that brackets W345 and W351, indicating that dsb2 formation is at least one factor that governs EFEMP1 secretion. These results are fully consistent with an enhanced ER folding environment at a permissive temperature for both WT and R345W EFEMP1 and thus more complete dsb2 formation in the ER, enabling enhanced proper folding and secretion.