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Acta Biochim Biophys
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doi:10.1111/j.1745-7270.2005.00080.x |
Intracellular Distribution, Assembly and Effect of Disease-associated Connexin 31 Mutants in HeLa Cells
Li-Qiang HE&, Yu LIU, Fang CAI, Zhi-Ping TAN, Qian PAN, De-Sheng LIANG, Zhi-Gao LONG, Ling-Qian WU, Liang-Qun HUANG, He-Ping DAI, Kun XIA*, Jia-Hui XIA, and Zhuo-Hua ZHANG
National Laboratory
of Medical Genetics, Central South University, Changsha 410078, China
Received: February
18, 2005
Accepted: April 30,
2005
This work was
supported by the grants from the National High Technology Research and
Development Program of China (No. 2002BA711A07-03, 08), the Major State Basic
Research Development Program of China (No. 2004CB518800) and the National
Natural Science Foundation of China (No. 31830200)
&
Present
address: 24#, Lane 1400, West Beijing Road, Medical Genetics Institute of
Shanghai Jiaotong University, Shanghai, China
*Corresponding
author: Tel, 86-731-4805357; Fax, 86-731-4478152; E-mail, [email protected]
Abstract Mutations in connexin 31 (Cx31) are associated with erythrokeratodermia variabilis (EKV), hearing impairment and peripheral neuropathy; however, the pathological mechanism of Cx31 mutants remains unknown. This study analyzed 11 disease-associated Cx31 variants and one non-disease-associated Cx31 variant and compared their intracellular distribution and assembly in HeLa cells and their effect on these cells. The fluorescent localization assay showed no gap junction plaque formation in the cells expressing the recessive EKV-associated mutant (L34P) and four hearing impairment-associated mutants (66delD, 141delI, R180X and E183K), significantly reduced plaque formation in the cells with five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) and no obvious change in the cells with two other mutants (I141V and 652del12). Immunoblotting analysis showed that 12 mutated Cx31s, like WT-Cx31, are able to form the Triton X-100 insoluble complex; however, the quantity of Triton X-100 insoluble complex in the transfected HeLa cells varied among different Cx31 mutants. Additionally, the expression of five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) caused cell death in HeLa cells. However, the five hearing impairment-associated mutants did not induce cell death. The above results suggest that disease-associated mutants gain deleterious functions differentially. In summary, disease-associated Cx31 mutants impair the formation of normal gap junctions at different levels, and the diseases associated with Cx31 mutations may result from the abnormal assembly, trafficking and metabolism of the Cx31 mutants.
Key words connexin 31; erythrokeratodermia variabilis; hearing impairment; peripheral neuropathy; gap junctional intercellular communication (GJIC)
Gap junctions consist of connexin (Cx) and mediate cell-cell communication via direct intercellular exchange of small molecules (<1 kDa). To date, 19 Cx genes have been found in the mouse genome and 20 Cx genes have been found in the human genome [1]. Generally, gap junctions are formed by homomeric or heteromeric hemichannels that are assembled by the same or different kinds of connexin [2]. Mutations in connexin have been identified with various inherited diseases, including Cx32 mutation in X-linked Charcot Marie tooth disease [3,4], Cx26 and Cx30 mutations in deafness and skin diseases [5-11], Cx46 and Cx50 mutations in hereditary cataracts [12-20] and Cx31 mutation in erythrokeratodermia variabilis (EKV) and hearing impairment with/without peripheral neuropathy [21-26].
Cx31 is an important member of the connexin family, but the
molecular mechanism of Cx31 in human diseases remains unclear. Diestel et al.
[27] reported that the Cx31 mutant (G12R) was expressed at a comparable level
as wild type Cx31 (WT-Cx31) and localized on the plasma membrane. It also
showed a higher conductance than WT-Cx31 in dye couple studies. Di et al.
[28] reported that four EKV-associated Cx31 mutants (G12R, G12D, R42P and C86S)
exhibited defective trafficking to the plasma membrane and that the
deafness/neuropathy-associated mutant 66delD had a primarily cytoplasmic
distribution, but certain proteins were visualized at the plasma membrane in a
few transfected cells. These findings suggest that the distributions of Cx31
mutants are different.
Cellular localization assays have indicated that many connexin mutants fail to assemble or localize to the cell membrane to establish normal gap junction intercellular communication (GJIC) [29,30]. Biochemical assays have also shown that many of the mutated connexins wrongly target the gap junctions and/or fail to oligomerize correctly into hemichannels [31].
In this study, the subcellular localization, effect on transfected cells and solubility in Triton X-100 of 12 Cx mutants in HeLa cells were analyzed. The study shows that different Cx31 mutants differ in terms of intracellular distribution, assembly and effect on HeLa cells.
Materials and Methods
Construct with chimeric Cx31 mutation/EGFP
The Cx31 mutants (G12R, G12D, L34P, R42P, 66delD, C86S, F137L, I141V, 141delI, R180X, E183K and 652del12) were produced by PCR using gene splicing by overlap extension (Table 1).
In the primary PCR, two segments of the Cx31 (C-segment and T-segment) were amplified from pEGFP-Cx31 using the primers Cx31-F and Cx31-R with the following conditions: 5 min at 95 ºC; 30 cycles of 20 s each at 95 ºC, 30 s at 50 ºC and 45 s at 72 ºC; and 10 min at 72 ºC. Moreover, Cx31 was produced using the primers Cx31-F/Cx31-R in the secondary PCR under the following conditions: 5 min at 95 ºC; 30 cycles of 20 s each at 95 ºC, 30 s at 62 ºC and 45 s at 72 ºC; and 10 min at 72 ºC. Furthermore, 12 mutants were generated by PCR using specific primers listed in Table 1, under these conditions: 5 min at 95 ºC; 30 cycles of 20 s each at 95 ºC, 30 s at 62 ºC and 45 s at 72 ºC; and 10 min at 72 ºC. Primers were synthesized by Shanghai Bioasia (Shanghai, China). After the amplification, the PCR products were directly cloned into a TA cloning vector, pGEM-T (Promega, Madison, USA). The mutated Cx31 fragments were cut with two restriction enzymes (EcoRI and SalI; TaKaRa, Dalian, China), and further cloned into the pEGFP-N1 vector (Clontech, Mountain View, USA). All mutants were sequenced, and the clones with correct base changes were chosen for subsequent study.
Transfection with Cx31/EGFP fusion constructs
HeLa cell line deficient in GJIC was purchased from CCTCC and maintained in Dulbecco's modified Eagle's medium, supplemented with 10% FBS (Gibco BRL, Gaithersburg, USA), 100 U/ml penicillin and 100 mg/ml streptomycin, at 37 ºC in a moist atmosphere containing 5% CO2. Transfection was carried out using Lipofectamine 2000 reagent (InvitrogenCarlsbad, USA) according to the manufacturer's instructions. Generally, a ratio of 1 mg DNA vs. 2 ml Lipofectamine 2000 was used for the HeLa cells. 24 h post-transfection, cells were harvested for Western blotting, or fixed with cold methanol for fluorescent staining. To select HeLa cell colonies stably expressing WT-Cx31 or Cx31 mutants, the selective medium containing 800 mg/ml G418 was renewed at 4-d interval. After 2-3 weeks, single cell colonies were obtained. Under the fluorescence microscope, the cell clones displaying green fluorescence were picked for further culture.
Immunofluorescent staining
For the fluorescent staining of endoplasmic reticulum (ER) or Golgi apparatus, HeLa cells were fixed with cold methanol for 15 min, washed 3 times with 0.1% Triton X-100/PBS, 10 min each time, and then stained with con A or WGA (conjugated with Alexa Fluor 594) for 15 min, and then washed 3 times with PBS. HeLa cells were observed using a fluorescence microscope, and images were taken using a laser-scanning confocal microscope (Bio-Rad Inc., Hercules, USA).
Solubility analysis of Cx31 mutants in Triton X-100 solution
24 h after transfection, HeLa cells expressing Cx31 mutants were rinsed once with PBS and incubated on ice for 30 min with PBS containing 1% Triton X-100 and a proteinase inhibitor cocktail (Sigma, St. Louis, USA). The cells were gathered by scraping, and then centrifuged at 100,000 g for 30 min. The insoluble fractions were lysed in SDS sample buffer (0.5 M Tris-HCl, pH 6.8, 20% glycerol, 4% SDS) and the protein concentration was determined using a Bio-Rad DC protein assay kit. Equal amounts of each sample were separated by 10% SDS-PAGE, and then transferred to a polyvinylidene fluoride (PVDF) membrane by electro-transfer. The PVDF membrane was incubated overnight with PBS containing 5% skimmed milk and 3% BSA. The primary antibody (rabbit anti-Cx31 or anti-GFP polyclonal antibody, 1:1000; Clontech) was then added for 2 h and washed 3 times with PBS with 0.1% Trition X-100 (PBST). Next, the secondary antibody (HRP-conjugated goat anti-rabbit antibody, 1:10,000; CalbiochemSan Diego, USA) was added for 1 h, and washed 3 times with PBST. The membrane was then detected using an ECL kit (Amersham Biosciences).
Results
Localization of the Cx31 mutants
Fig. 1 showed the cellular localization of WT-Cx31 and 12 Cx31 mutants 24 h post-transfection. HeLa cells expressing EGFP exhibited green fluorescence in whole cells, both in the cytoplasm and nucleus [Fig. 1(A)]. HeLa cells expressing WT-Cx31/EGFP displayed punctate staining and aggregation at the plasma membrane, particularly in the regions of cell-ell contact [Fig. 1(B)]. Punctate staining and aggregation at the plasma membrane were also observed in G12R, G12D, R42P, C86S, F137L, I141V and 652del12, particularly in the regions of cell-cell contact [Fig. 1(C-I)]. In L34P, 66delD, 141delI, R180X and E183K strains, punctate staining and aggregation did not exhibit at the plasma membrane [Fig. 1(J-N)] although Cx proteins existed in the cytoplasm, mainly in ER or Golgi apparatus.
The number of cells forming gap junction channels in adjacent HeLa cells both expressing Cx31 mutants was also analyzed. The analytical results indicated that the portion of cells with gap junction plaque in the five dominant EKV mutants (G12R, G12D, R42P, C86S and F137L) was significantly lower compared with that of WT-Cx31 (P<0.05), while the proportions in the two mutants (I141V and 652del12) clearly did not decrease compared with WT-Cx31 (P>0.05) (Fig. 2).
In G12R, G12D, R42P, C86S and F137L strains, cell and nuclei morphology changed 24 h post-transfection. G12D and F137L showed similar patterns (Fig. 3), and G12R, R42P and C86S were also similar (data not shown), which is consistent with the results of Common et al. [30]. Furthermore, the recessive EKV mutant, L34P, was found to be not lethal to HeLa cells.
Using the G418 screening, HeLa cell lines that stably expressed WT-Cx31, L34P, 66delD, R180X, E183K, I141V, 141delI and 652del12 were obtained (Fig. 4), but HeLa cell lines that stably expressed G12R, G12D, R42P, C86S or F137L could not be obtained, which is contrary to the conclusion of Common et al. [30] that "defective trafficking and cell death is characteristic of skin disease-associated connexin 31 mutations".
Solubility of Cx31 mutants in Triton X-100 solution
The formation of oligomers is a key step in establishing gap junctions at the cell surface. Previous studies have shown that connexin oligomers are insoluble in 1% Triton X-100, but its monomer is soluble [32]. This study examined whether the Cx31 mutants could form insoluble oligomers in 1% Triton X-100. The results revealed that all these mutants could form insoluble oligomers in 1% Triton X-100 solution (Fig. 5). However, the number of oligomers formed differed among the Cx31 mutants. Fewer oligomers were formed by R180X than those of other mutants.
Discussion
Connexin, an essential component of gap junctions, must be trafficked to the cell membrane to execute its biological function. Cellular localization and function assay suggest that mutations in connexin cause degradation in their expression, assembly, trafficking or formation of functional gap junctions, thereby damaging communication between neighboring cells.
Deschenes et al. [29] studied the cellular localization of nine X-linked Charcot Marie tooth disease (CMTX)-associated Cx32 mutants in PC12J cells. These Cx32 mutants were grouped into three classes: (1) mutant mRNA was transcribed, but little or no protein was detected; (2) mutant protein was detectable in the cytoplasm and at the cell surface, where it appeared as plaques and punctate staining; (3) the immunoreactivity of the mutant protein was restricted to the cytoplasm and frequently colocalized with the Golgi apparatus. Common et al. [30] studied four Cx30 mutants, and found that three skin disease-associated mutants failed to be trafficked to the plasma membrane, and thus could not form functional gap junctions. The deafness-associated mutant can be trafficked to the membrane, but has no channel activity.
The present study examined 11 disease-associated Cx31 mutants in HeLa cells. Three types of mutations, according to subcellular distribution, were observed. Type I, including L34P, 66delD, 141delI, R180X and E183K, is characterized by the cytoplasmic accumulation of Cx31 and the absence of cell surface expression. These mutants alter the trafficking so that the proteins accumulate in intracellular compartments, such as Golgi apparatus or other structures like ER. Type II includes five dominant EKV Cx31 mutants, G12R, G12D, R42P, C86S and F137L. The expression product of these mutants was partially trafficked to the cell surface, so they are lethal to HeLa cells. Type III is represented by I141V. I141V migrates mainly to the cell surface, which resembles that of WT-Cx31. These findings suggest that different mutations in Cx31 exhibit different subcellular distributions and none can form functional gap junction intercellular channels.
Mutations in the plasma membrane or secreted proteins that inhibit transport to the cell surface might cause disease by general mechanisms [33,34]: first, the affected protein can not be normally transported to the plasma membrane, but can be routinely degraded; second, the mutant can not be degraded, and thus accumulates within the cell and induces chronic endoplasmic reticulum stress responses, causing major changes in cell physiology, such as apoptosis, abnormal differentiation, altered proliferation, and so on. In this study, Type I Cx31 mutations do not induce chronic endoplasmic reticulum stress responses as stable cell lines were obtained. Therefore, these mutants may cause disease via the first mechanism. Although Type II Cx31 mutants can be trafficked to the cell membrane and form gap junctions, their function is abnormal because their expression can cause cell death. Therefore, the disease may result from the abnormal Cx31 function. Type III I141V mutant is found to coexist in the allele with 141delI mutant [25]. Therefore, this mutant may be recessive, and may cause a defect in normal GJIC. However, further studies will be necessary to confirm this hypothesis.
Notably, this study showed that four EKV-associated mutants (G12R, G12D, R42P and C86S) could be trafficked to the plasma membrane and exhibit punctate staining; however, another mutant (66delD) could not be visualized at the plasma membrane, in contrast to the findings of Di et al. [28]. We believe that these differences can be explained as follows.
(1) Connexin expression may be dependent on the type of cell. Cx31 mutants were transfected into NEB1 cells by Di et al. [28], while Cx31 mutants were transfected into HeLa cells in this study. Owing to connexin protein deficiency, HeLa cells have been widely used to study connexin functions [35-42]. NEB1 cells, as a kind of keratinocytes, express several types of connexin. Endogenous connexins may affect the expression of transfected Cx31. However, Cx31 should be expressed in the epidermis, which contains several connexins. Therefore, the subcellular localization of Cx31 mutants in NEB1 cells may resemble the actual distribution of Cx31 mutants in patients more closely than the localization in HeLa cells. The immunolocalization of Cx31 mutants in patients provides further evidence of this.
(2) Cx31 mutants were introduced into mammalian cells via different methods. Diestel et al. [27] constructed G12R into an inducible vector and transfected them into HeLa cells via calcium phosphate crystals. Furthermore, Di et al. [28] constructed Cx31 mutants into pEGFP-N3, and microinjected them into NEB1 cells. In this study, Cx31 mutants were constructed into pEGFP-N1, and transfected them into HeLa cells by Lipofectamine 2000.
This study also investigated the border between the transmembrane
and cytoplasmic domain of Cx31 using NCBI or TMpred software. Although the
predicted positions of the transmembrane and extracellular and intracellular
domains differ among amino acid groups, the locations of the mutated sites
remain consistent. Fig. 6 shows the positions of these sites.
Disease-associated mutations were distributed in the whole structure of Cx31
except the IC2 and TM4 domains, indicating that the domains of Cx31 may play
different roles in the physiological functions of Cx31. The C-terminal part of
Cx31 may play a role in Cx31 oligomer formation in the cells, as the R180X
mutant transfected cells contain fewer oligomers than other mutants and
WT-Cx31.
In summary, this study has shown that different disease-associated Cx31 mutants exhibit different subcellular distributions, abilities in the formation of oligomers and effects on transfected HeLa cells, suggesting that diseases associated with Cx31 mutations may result from the abnormal assembly and trafficking of the mutants. Furthermore, this study has shown that deafness-associated mutations and skin disease-associated mutations have different influence on the function of Cx31. These findings may be helpful in understanding the mechanism of diseases caused by Cx31 mutations.
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