| Matrix metalloproteinases,also called MMPs,is a hydrolase superfamily which could degrade extracellular matrix and need Ca2+ and Zn2+ to be its cofactors.MMPs take part in many important physiological functions in human beings,including tissue remodeling,wound repairing,bone growth and embryonic development.So far,25 different MMPs have been found in human body,they could be separated into 6 groups according to their substrate specificity.They are collagenase,gelatinase,stromelysin,matrilysin,membrane-type MMPs and furin-activated MMPs.MMP-26 is a latest found member of the MMPs superfamily.It was first found and identified in human endometrial tumor cells in 2000.MMP-26 consists of three major domains,signal peptide(1-17),pro-peptide(18-89)and catalytic domain(90-261).Within its pro-peptide domain,there is a unique “Cysteine switch”,PH81 CGVPDGSD,the arginine in 81 of other MMPs was mutated into histidine in the sequence of MMP-26.MMP-26 might have an unconventional self-activation pathway.In the superfamily of MMPs,MMP-26 and MMP-7 have the highest degree of homology,39%,they both belong to the matrilysin sub-family.However,they possess some different properties.For instance,MMP-7 is a typical secreted protein,and it could bind to cholesterol sulfate in cell membranes,its substrates include fibronectin,laminin,and gelatin.MMP-26 has a signal peptide like MMP-7,but some researches prove that MMP-26 expressed mainly within human breast cancer cells and prostate cancer cells,and they couldn't find expression of MMP-26 in the culture supernatant.This means MMP-26 may have a different subcellular localization compared to MMP-7.Based on Doctor Wang Zhiyong's former research,MMP-26 could localize in the endoplasmic reticulum(ER),and through a series of site mutation and sequence substitution,he confirmed 80-125 region between pro-peptide and catalytic domain is the vital sequence for MMP-26 ER retention.In this article,through overlapping PCR method,we generated a series of MMP-26 truncated mutants,then fused it into p EGFP-N1.We made further efforts to confirm that 88-123 region of MMP-26 is the key sequence for MMP-26 ER localization.Furtherly,we swapped 93-128 region of MMP-7 for 88-123 region of MMP-26,we named it Mut-MMP-7.We found Mut-MMP-7 could also localize in the ER,this means 88-123 region of MMP-26 is a novel ER retention signal.In the former researches,we found the molecular size of MMP-26-GFP is larger than its theoretical size.According the N-glycosylation prediction results of MMP-26,we deduced that there might be N-glycosylation in MMP-26,the shift might be the molecular size of N-glycans.Thus,first we inhibited N-glycosylation of MMP-26 by tunicamycin(inhibitor of N-glycan synthesis)and deglycosylase(PNGase F,Endo H),and after these treatments,the molecular size of MMP-26-GFP was equal to the theoretical size.Then by site-directed mutagenesis,we generated MMP-26(N64A,N221 A and N64A/N221A).The molecular size of these three mutants were equal to loss of one or two N-glycan groups.Then by transient transfection,we found loss of N-glycosylation didn't interrupt ER retention of MMP-26.And this result does not conflict with our former results.According to Doctor Zhang Jinrui's former research,she has verified that MMP-26 and molecular chaperone GRP78 could co-locate in the ER and interact with each other,and MMP-26 could lower the expression level of GRP78 within the cell.In this paper,we identified that inhibition of N-glycosylation of MMP-26 impairs the interaction between MMP-26 and GRP78.We tried to investigate how MMP-26 interact with GRP78 intracellularly.We generated two kinds of MMP-26 stable transfection cells: MMP-26 knock-out and MMP-26 overexpression.Firstly,we studied how MMP-26 could influence cellular behavior of He La cells.Wo found that overexpression of MMP-26 in He La cells lower the proliferation and invasion ability of He La cells.Then we examined the expression level of MMP-26 and GRP78 in He La cells.When MMP-26 level was high,GRP78 was down-regulated,and when MMP-26 level is down,GRP78 tend to be up-regulated.Thus,we suppose that MMP-26 could down-regulated the expression of GRP78.GRP78 is a typical ER resident protein,it binds to three unfolded protein response sensor protein: IRE1,PERK and ATF6.We found when MMP-26 was over-expressed,expression of PERK was higher than usual,and so is CHOP protein.Since CHOP protein is a key molecule in the cell apoptosis pathway.We deduce that MMP-26 could promote apoptosis of He La cells.In summary,in this article,we confirmed 88-123 region of MMP-26 is the key sequence for its ER retention,this is a novel non-KDEL ER localization signal.And MMP-26 is an N-glycosylated protein and it has two N-glycosylation sites,N64 and N221.MMP-26 could weaken the proliferation and invasion ability of He La cells.According to Doctor Zhang Jinrui's research,we have verified again that expression of GRP78 was lowered by MMP-26 and this function is caused by unfolded protein response within the He La cells.Our research lay the foundations for intracellular functions of MMP-26. |
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