【作者】 彭志红；

【导师】 房殿春；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 背景和目的:在以前的研究中,我们证实rMuc3羧基端于合成早期即在内质网内发生了酶切,酶切位点位于LSKGSIVV基序的GS位点。30kDa的氨基端片段并没有脱落,而是与49kDa的羧基端片段以SDS或热敏感的非共价键连接在一起共同锚定在细胞膜上。酶切以及酶切后片段相互连接的生物学意义目前尚不清楚,可能有利于后续可溶性的细胞外区域释放至肠腔。我们同时发现rMuc3羧基端SEA组件的酶切需要LSKGSIVV基序后续较远肽段的参与,片段的连接需要完整的SEA组件,而与N型糖基化无关。进一步我们发现,49kDa的膜锚定片段经历了二次酶切,产生30kDa片段,并经Western blotting,免疫沉淀和N-glycosidase F脱糖基化检测和证实了酶切产物。两次酶切的发生可能有利于Muc3在肠上皮细胞表面形成可分泌形式和(或)参与肠上皮细胞表面受体、配体的识别进而参与体内细胞信号转导。但LSKGSIVV基序的酶切是蛋白酶介导的酶切还是自酶切,目前尚不清楚。探索酶切的机制对于rMuc3及其他包含SEA组件的蛋白质有非常重要的意义。最近证实,MUC1内SEA组件的酶切是自酶切,并进一步阐明了其酶切的机制。但MUC1内SEA组件的104个氨基酸残基与rMuc3SEA组件174个氨基酸残基同源性非常低(仅6.9%),因此,MUC1SEA组件的酶切方式不能类推至rMuc3等其他粘蛋白。本课题即是进一步证实我们以前发现的rMuc3羧基端SEA组件LSKGSIVV基序的酶切方式,并探索这种蛋白质翻译后修饰发生的生物学意义。方法:1、含目的蛋白原核表达载体的构建、表达在以前的研究中,我们已构建了含rMuc3羧基端381个氨基酸的p20、p20G/A真核表达载体。在本实验中我们以p20、p20G/A质粒为模板,采用PCR方法获得含rMuc3羧基端381个氨基酸目的片段,将此片段插入N端含His标签的pQE30原核表达载体中,命名为pQE30-Muc3和pQE30-Muc3(g/a)。将pQE30-Muc3和pQE30-Muc3(g/a)转化宿主菌M15,重组菌在含100mg/L氨苄青霉素和50 mg/L卡那霉素的LB培养基中加入IPTG (终浓度1Mm)37℃诱导获得目的蛋白表达,Western blotting检测表达蛋白,以在原核细胞水平探讨大鼠Muc3分子羧基端SEA组件的酶切情况。2、pQE30-Muc3(g/a)表达蛋白的纯化和体外孵育pQE30-Muc3(g/a)表达蛋白采用Ni-NTA agarose纯化。将转化pQE30-Muc3(g/a)的重组菌诱导后超声破菌,超声上清与Ni-NTA agarose在4℃摇床上共孵育2小时,上柱,蛋白检测仪监测纯化过程,依次用裂解缓冲液、洗涤缓冲液1、洗涤缓冲液2洗柱,最后用洗脱缓冲液洗脱,从而获得目的表达蛋白。分别取纯化蛋白在37℃水浴孵育4、8、16、24、36和48小时,Western blotting检测孵育蛋白,观察纯化蛋白孵育后的酶切情况。3、SEA组件的完整性对酶切发生的影响利用ClustalX对多个含SEA组件的蛋白质进行序列比对,获得p20t后续保守的氨基酸残基位点,分别为174位丝氨酸,201位半胱氨酸,212位酪氨酸和223位酪氨酸。采用定点突变技术,设计相应突变引物,以p20SEA为模板,基于PCR扩增得到不同含终止密码子的不完整SEA组件突变体,并经测序验证后,利用阳离子脂质体Lipofectamine2000将各突变体及p20、p20SEA瞬时转染入COS-7细胞中,通过Western blotting检测各突变体的酶切发生情况,以发现新的、未证实的SEA后续79个氨基酸中对构象的维持及酶切的发生起关键作用的多肽序列。4、rMuc3SEA组件的分子模建人MUC1和CA125(鼠muc16的同系物)SEA组件的晶体结构已通过NMR获得,rMuc3SEA组件与二者有一定的同源性,利用生物信息学知识,运用SGI insightII工作站中的homology模块对rMuc3SEA组件进行同源性模建,获得rMuc3羧基端SEA组件的3D结构图,从结构上进一步分析SEA组件内酶切发生的结构基础,丰富SEA组件酶切方式的物质内涵。5、将不同酶切状况的真核表达载体稳定转染Lovo细胞,转染及表达鉴定后流式细胞仪观察转染后细胞G2/M比例及凋亡情况,划痕实验观察细胞在机械性损伤后迁移情况,Transwell小室实验观察细胞侵袭能力,以初步探讨酶切发生的可能的生物学意义。结果:1、获得pQE30-Muc3和pQE30-Muc3(g/a)原核表达载体,转化宿主菌,重组菌在37℃、IPTG终浓度为1mM诱导条件下可见目的蛋白表达,Western blotting检测发现表达产物中包括55kDa的全长的rMuc3羧基端分子,该分子同时可以被V5和Myc抗体所识别;49kDa的仅能被Myc抗体所识别,但不能被V5抗体所识别的酶切后的羧基端片段;30kDa的仅能被V5抗体所识别,但不能被Myc抗体所识别的酶切后的氨基端片段。这表明在细菌中rMuc3羧基端也如真核细胞中一样发生了酶切。一般来说,细菌中不可能存在能酶切真核细胞中表达而细菌中不表达的rMuc3蛋白羧基端的特异性蛋白酶。这一研究为rMuc3SEA组件的酶切是自酶切这一假设提供了初步证据。2、pQE30-Muc3(g/a)原核表达蛋白纯化后37℃水浴孵育,Western blotting时用N端V5抗体检测发现在4小时、8小时只可见大小约55kDa的全长片段,16小时可见大小约55kDa的全长片段减少而30kDa酶切后片段明显增加,随着孵育时间的延长,全长片段未检测到,纯化蛋白仅为30kDa的酶切后片段。这个发现更让我们肯定rMuc3SEA组件在无其他外源性蛋白的影响下发生进一步酶切。随着孵育时间的延长,N端30kDa片段是唯一的裂解片段,排除了rMuc3羧基端纯化蛋白降解的可能。因此,我们认为rMuc3羧基端SEA组件的酶切是自酶切。3、将研究的rMuc3羧基端保守的第174位丝氨酸、201位半胱氨酸、212位酪氨酸、223位酪氨酸分别突变为终止密码子后,瞬时转染COS-7细胞,产生一组截短的rMuc3羧基端蛋白并分泌至培养基中。培养上清行N端V5抗体Western blotting检测,可见在201位半胱氨酸、212位酪氨酸、223位酪氨酸突变后均可见大小约30kDa的酶切后片段。因此推测174-201的氨基酸序列对维持SEA组件的正常构象非常重要,从而保证rMuc3SEA组件自酶切的发生。4、模建的结构提示rMuc3SEA组件是由四个α螺旋和四个β片层组成的结构域,没有无规卷曲,与人MUC1SEA组件的晶体结构非常相似,酶切基序位于β2和β3之间的转角处。SEA组件内174S至201C的氨基酸序列位于α4与β4形成的转角上,且在空间上与酶切基序的位置非常靠近。可能这正是174S至201C的氨基酸序列影响自酶切的原因所在。rMuc3羧基端SEA组件的分子模建为我们提供了理解rMuc3SEA组件自酶切发生的结构信息。5、rMuc3羧基端稳定转染人Lovo细胞(该细胞表达截短形式的MUC3,缺乏MUC3的胞浆尾部,因此Lovo细胞中MUC3的功能肯定受影响)后,流式细胞仪检测显示,Lovo/p20组进入G0/G1期细胞减少,而进入S期和G2/M期的细胞较其他组明显增加,差异有统计学意义(p<0.05), Lovo/p20G/A组、Lovo/p20S/A组、Lovo/pSec及未转染组之间无明显差异(p>0.1);Lovo/p20组穿越Matrigel“屏障”浸润的细胞数显著增加,与Lovo/p20G/A、Lovo/p20S/A、Lovo/pSec及未转染组相比差异有统计学意义(p<0.05),而Lovo/p20S/A、Lovo/pSec及未转染组之间差异不明显(p>0.1);细胞划痕实验结果显示, Lovo/p20组培养24 h时,细胞向“伤口”迁移的细胞数多于Lovo/p20G/A, Lovo/p20S/A, Lovo/pSec及未转染组,Lovo/p20组1. 9±0.63与Lovo/p20G/A组(1. 16±0.41), Lovo/p20S/A组(0.85±0.41),Lovo/pSec组(1.15±0.44)组相比差异有统计学意义(P<0. 05),Lovo/p20G/A组、Lovo/p20S/A组、Lovo/pSec组及未转染组之间无明显差异(p>0.1)。这些发现提示rMuc3SEA组件的自酶切可能掌控该蛋白功能的发挥。结论:1.在原核细胞表达水平和原核表达蛋白纯化后体外孵育水平均证实大鼠Muc3分子羧基端SEA组件发生酶切,结合前期在真核水平的研究结果和酶切位点的结构特点,表明该酶切的发生是没有蛋白酶介导的自酶切的结果。2.应用定点突变技术研究SEA组件后续79个氨基酸对酶切的影响时发现羧基端174位丝氨酸至201位半胱氨酸之间的氨基酸序列对于酶切的发生非常重要,可能在维持SEA组件的正常三维结构起关键性的作用,从而触发酶切的发生。3.rMuc3SEA组件蛋白质分子模建的发现从结构上为我们提供了rMuc3SEA组件自酶切发生的必然性。4.体外实验发现,rMuc3SEA组件的自酶切可能掌控其功能的发挥,在体外实验中能促进细胞增生,增强细胞迁移和侵袭,从而影响细胞的生物学行为。更多还原

【Abstract】 Background and objectives:In a previous paper, we presented evidence that the expressed C-terminal domain of the rodent membrane mucin Muc3 (construct p20) undergoes proteolytic cleavage between the glycine and serine within the LSKGSIVV amino acid sequence during an early period of biosynthesis in the endoplasmic reticulum (ER). The 30 kDa N-terminal cleavage fragment is not secreted, but remains associated with the 49 kDa C-terminal membrane tethered fragment by non-covalent disulphide bond-independent interactions. The biological purposes of the cleavage and the association of the fragments are not understood, but are possibly important for the later release of the soluble extracellular domain into the intestinal lumen. Then, we found that cleavage within the SEA module (sea-urchin sperm protein, enterokinase and agrin module) of rat Muc3 requires participation of peptide sequences located C-terminal of and distant from the LSKGSIVV cleavage site, and association of the fragments requires the SEA module, but is independent of N-linked oligosaccharides. Further, we showed that the 49 kDa membrane-anchored fragment undergoes a further cleavage reaction, which decreases its size to 30 kDa. Western blotting, pulse–chase metabolic incubations, immunoprecipitation and deglycosylation with N-glycosidase F were used to detect and identify the proteolytic products. Both the first and second cleavages are presumed to facilitate solubilization of Muc3 at the apical surface of enterocytes and/or enhance the potential for Muc3 to participate in ligand–receptor and signal transduction events for enterocyte function in vivo. But it is still unclear how the proteolytic cleavage in the LSKGSIVV motif occurred, whether it was caused by protease or autoproteolysis. This kind of data is important to the SEA module-containing protein. Recently, the cleavage within the SEA module of human MUC1 was identified as an autocatalytic reaction, and the mechanism for this cleavage was elucidated However, the sequence similarity between the 104 amino acids that constitute the human MUC1 SEA module and the 174 residues of the rat Muc3 SEA module is only 6.9% (12/174). Due to the lack of sequence homology between the MUC1 SEA module and the rat Muc3 SEA module, the finding of the cleavage reaction in the MUC1 SEA module cannot be extrapolated to other mucins. This study was designed to demonstrate the mechanisms related to our previously described proteolysis within the SEA module of rodent Muc3 and decipher its rationale of this kind of posttranslatinal modification.Methods:1.Expression of the interested protein: DNA encoding residus of 381 amino acids within carboxyl terminal domain of rodent Muc3 obtained by PCR templated from p20 and p20G/A which were described previously(ref. 26 ) were inserted into the pQE30 prokaryotic expression vector with a coding for an N-terminal 6×His tag and the construction of prokaryotic expression of the rodent Muc3 carboxy-terminal domain was designated as pQE30-Muc3 and pQE30-Muc3(g/a). M15 cells were transfected by the pQE30-Muc3 or pQE30-Muc3(g/a). Cells were cultured in LB medium with 100mg/L Ampicilin and 50 mg/L kanamycin and induced at OD600=0.5 with 1mM IPTG and continued for 6h at 37℃.The product from pQE30-Muc3(g/a) transfected bacteria was purified by Ni-NTA agarose and the purified protein was incubated in 37℃for 4, 8, 16, 24, 36 and 48 hours in PBS separately.The incubated protein was subject to SDS/PAGE (12%) and detected by anti-V5 or anti-Myc antibodies (anti-V5, 1:2500; anti-Myc, 1:1000).2.Site-directed mutation: Sequence alignment of the SEA modules was performed by ClustalX. The conserved amino-acid residues were 174th serine,201th thioserine,212th tyrosine and 223th tyrosine, then mutated to stop coden by PCR templated with p20SEA to produce a series of new constructions which produced truncated forms of rMuc3. COS-7 cells were transfected transiently with these plasmids to produce different truncated SEA module of rodent Muc3 in order to find new and unidentified polypeptide sequences which located at the 79 amino acids, C-terminal end of SEA module of rMuc3 and were critical to keep its proper conformation and the occurrence of autoproteolysis.3.Molecular Modelling: The molecular modelling of rMuc3 SEA module was performed in Homology Model Block of SGI Insight II software package, SWISS MODEL WORKSPAC based on the solution structures of SEA domain from the murine homologue of ovarian antigen CA125 (MUC16) and membrane-bound MUC1 mucin identified by NMR.4.Cell culture and transfection experiments: Lovo cells (Shanghai Academia Sinica Life Science Research Institute) were cultured in F12K supplemented with 10% (v/v) FBS. cells were seeded into 3.5-cm-diameter tissue-culture dishes at a density of approx. 8×106 cells/dish. At 40-50% confluence, DNA transfections in cells were carried out with 2μg of plasmid and 10μl of LIPOFECT2000 in the presence of F12K without FBS. pSecTag2 transfection served as a control in each experiment. The hygromycin -resistant colonies were isolated by the ring cloning method, expanded, and maintained in medium supplemented with 100ug/mL hygromycin .PCR,Western blotting and membrane-targetting experiments were to identify the successful transfection.5.Cell Cycle Analysis: Lovo cells were transfected with p20, p20G/A, p20S/A or the empty vector, pSec, respectively. The transfected cells were incubated at 37°C, 5%CO2 for 48h. Cells were washed once in PBS, fixed in 90% Methanol, and stained in PI buffer containing Propidium Iodide, RNase A (Invitrogen), 0.1% Triton X-100 in PBS. Analysis was performed on flow cytometry and G2/G1 ratios were calculated.6.Cell migration assay: Lovo cells grown to 90%-100% confluency in 24-well plates were cultured overnight in serum-free medium. The medium was replaced with PBS, and the monolayers were wounded mechanically using a steriled transferpettor tip. After wounding, cells were rinsed twice with PBS and further incubated in F12K medium without serum for 24h at 37℃, 5%CO2. Those cells that had migrated from the wounded edge were counted at 100×, using an inverted light microscope. Five successive fields were counted and averaged within 3 well.The assay was repeated three times.8.Cell invasion assay: Motility and invasion capability in vitro were measured in transwells chambers assay. 100μl diluted Matrigel gel solution was put into upper chambers of the transwell inserts (6.5 mm, 8μm pore size; Costar Inc., USA). Incubated the inserts at 37°C for 4 h for gelling and then pretreated with serum-free medium at 37°C for 1 h before seeding cell at a density of 1×105 per well in 100μl medium with 1% FBS. The lower chambers of the transwells were filled with 500μl medium containing 10% FBS. The transwells were then incubated at 37°C with 5% CO2 for 24 h to allow cells to migrate. At the end of incubation, the cells on the upper side of the insert filter were completely removed by wiping with cotton swab. Cells that had invaded through the Matrigel-coated filter were stained with HE. Cells that had invaded the Matrigel and reached the lower surface of the filter were counted under a inverted microscope of 200×. Five fields of vision were chosen and the numbers of the invaded cells at the lower surface of the filter were counted, and the results from three separate chambers were then averaged. The assay was performed in triplicate.Results:1. The carboxy-terminal domain of rodent Muc3 were expressed in E. coli at 37℃induced by IPTG at 100mmol/l. The products contained the 55kDa, full-length, carboxy-terminal domain of rodent Muc3 recognized by both anti-V5 and anti-Myc antibodies, 49 kDa C-terminal fragment recognized only by anti-Myc antibody, but not by anti-V5 antibody, and 30kDa N-terminal fragment recognized only by anti-V5 antibody, but not by anti-Myc antibody, which indicated that the carboxy-terminal domain of rodent Muc3 was cleaved in bacteria as same as in the eukaryotic cells. Based on the general knowledge, it is impossible for the bacteria to have the specific protease to cleave the carboxy-terminal domain of rodent Muc3, a protein present in rat, not in the bacteria. This data provides a primary evidence to the autoproteolysis of the SEA module of the rMuc3.2. Products from pQE30-Muc3(g/a) was purified and incubated in PBS at 37℃, then deteced by Western blot with anti-V5 antibody. The main products after 4h or 8h incubation were the 55kDa full length one. After 16h incubation, the 55kDa full-length products decreased and the cleaved 30kd N-terminal fragment increased dramatically. With further incubation, the full-length products disappeared, only the 30kd N-terminal fragment existed. This data confirmed that the carboxy-terminal domain of rodent Muc3 was undergone the further cleavage without any other protein influence. The N-terminal 30 kDa fragment was the only cleaved fragment during the different time of incubation, it excluded the possibility of the degradation of the purified carboxy-terminal domain of rodent Muc3 from eukaryotic cells. So we concluded the cleavage in the SEA module in the carboxy-terminal domain of rodent Muc3 was based on the autoproteolysis.3. The conserved amino acids including S174、C201、Y212、Y223 in the SEA module were mutated to stop coden, respectively, and then COS-7 cells were transfected transiently with a series of mutated plasmids which produced a truncated forms of rMuc3 carboxyl terminal domain and secreted into the spent media. The spent media were detected by Western blotting with anti-V5 antibody. The cleaved 30kd N-terminal fragments were detected in 201C、212Y、223Y mutated constructs. So the amino acid sequence between 174 and 201 is critical to keep the proper comformation of the SEA module of rMuc3 and guarantes the occurrence of the autoproteolysis of the SEA module of rMuc3.4. The SEA module consists of a four-stranded antiparallelβ-sheets and fourα-helices occurring in the order ofβ1→α1→α2→β2→β3→α3→α4→β4 and is similar to that of human MUC1. The cleavage site is located in the turn betweenβ2 andβ3.The sequence between 174S and 201C resides in the turn formed byα4 andβ4, and is near to the site of cleavage. It is the possible reason for the sequence between 174S and 201C resides to affect the autoproteolysis. The molecular modelling of the SEA module of rMuc3 provides a structural information to understand the autoproteolysis of the SEA module cleavage.5. The introduction of the carboxy-terminal domain of rodent Muc3 into the Lovo cells (which had a different splicing variant of MUC3 and no cytoplasmic tail of MUC3, and definitely affected the function of MUC3 in Lovo cells) drove more cells into the G2/M phase than the other groups measured by FACS (p<0,05); Cells transfected with p20 showed an evident increase in cell migration and invasion over 24h compared with cells transfected with p20G/A、p20S/A and pSec vecor or non-transfected. The data indicated that the autoproteolysis of the SEA module of rMuc3 controlled its function.Conclusion:1. Our studies indicate that the SEA module within carboxyl terminal domain of rodent Muc3 undergoes autoproteolysis.2. The amino-acid residues between 174S and 201C may be critical to for the autoproteolysis.3. The autoproteolysis of the SEA module of rMuc3 determined its functional composition. The autoproteolytic rMuc3 C-terminal domain may mediate cell proliferation, stimulates cell migration and modulates cell invasion in vitro.更多还原