A Study On How GMPPA/GMPPB Complex Regulate Human GDP-mannose Homeostasis And How GMPPB Mutation Result In Muscular Dystrophy

Muscular dystrophy is a disease with slow progress,poor prognosis and high disability rate.The symptoms of muscular dystrophy include muscle atrophy and slow-onset progressive symmetrical muscle weakness.Up till now,there are few effective treatments for muscular dystrophy.More studies on its diagnosis and treatment are in urgent need for patients to not only improve their life quality,but increase their survival rate as well.Previous studies have shown that mutations in GMPPB(GDP-mannose pyrophosphorylase B),which lead to defective nutrient metabolism in the glycosylation pathway,are linked with muscular dystrophy.During metabolism process,GMPPB reversibly catalyzes the formation of GDP-mannose,using GTP and mannose-1-phosphate as substrates.In bacteria,GMPPB functions as a monomer,while in mammalians it forms a close complex with GMPPA(GDP-mannose pyrophosphorylase A).GMPPA is the homologue of GMPPB.However,the functional molecular mechanism of GMPPB is poorly understood.Moreover,the functional molecular mechanism of GMPPA/GMPPB complex also needs to be elucidated.Mannose and its derivatives,including GDP-mannose,are critical for both bacteria and eukaryotes.In bacteria,GDP-mannose is necessary for the formation of capsular and other cell wall components.In eukaryotes,GDP-mannose plays a central role in protein glycosylation.However,GMPPA mutations lead to elevated GDP-mannose levels in patient lymphoblast.these patients also demonstrated abnormal brain and muscle development.Thus,maintaining proper GDP-mannose levels seems critical for human physiology,and aberrant cellular GDP-mannose levels have been associated with multiple human diseases.In summary,there is no relevant report on whether and how GMPPA/GMPPB regulated the concentration of GDP-mannose.In addition,the function of GMPPA is little known.To our best knowledge,there is no report on the structure of human GMPPB or GMPPA/GMPPB complex.Moreover,the underlying molecular mechanism needs to be elucidated.The objective of this study is as follows:(1)to obtain the structure of GMPPA/GMPPB complex by cryo-electron microscope;(2)to explore the function of GMPPA/GMPPB complex;(3)to investigate the role of GMPPA in regulating the concentration of GDP-mannose;(4)to explore the function of GMPPB in muscular dystrophy.Through exploring the expression and purification conditions,the GMPPA/GMPPB complex proteins were successfully expressed in Sf 9 insect cells.The structure of the complex was successfully obtained by cryo-electron microscope.We used Pymol,DALI and PISA software to analyze the structure of the GMPPA/GMPPB complex and find the key functional sites.We obtained the plasmids of wild type and mutants of human GMPPB gene through molecular cloning technology.We used E.coli expression system to obtain soluble GMPPB recombinant protein by changing expression strains,expression vector or induction temperature.After conducting the enzymatic reaction of human GMPPB wild-type protein and its mutant proteins,the concentration of GTP and GDP was determined by chromatographic method.Based on the above results,we compared the difference of their enzyme activity.Enzyme activity test and fluorescence spectrophotometer were used to verify the newly discovered functional sites.Then,correlation analysis of enzyme activity and clinical patient symptom scores was performed.This study discovered a child with congenital muscular dystrophy.The whole exon sequencing proved that this patient had a compound heterozygous mutation in the GMPPB gene(G214S and V111G).These compound heterozygous mutation sites in the GMPPB gene(G214S and V111G)were highly conserved in different species.The GMPPA/GMPPB complex was expressed and purified in an insect cell system,and the structural information of GMPPA/GMPPB complex in three conditions was obtained by cryo-electron microscope.The structure observation and comparison analysis found that the key sites of GMPPA and GMPPB combining with GDP-mannose were E85,Q247,and E80,D218.The obvious modification of GMPPA’s A Loop(358-380)was noticed in different conformational structures.The function of GMPPA/GMPPB complex was verified by enzyme activity.GMPPA/GMPPB complex had lower enzyme activity than GMPPB.It was found that GMPPA had no enzyme activity.The enzyme activity recovery test was carried out by simulating GMPPB.It turned out that the enzyme activity of mutant GMPPA Q247 D and truncated A Loop was not recovered.It was showed that GMPPA may play a negative regulatory function.The molecular mechanism of GMPPB was verified by enzyme activity.The enzyme activity of GMPPB was lost by mutating the key site of D218 Q.The key site of GMPPB enzyme activity was discovered for the first time.The key sites of the GMPPA/GMPPB complex was verified by enzyme activity.It was found that the enzyme activities of the three interface mutants in GMPPA and GMPPB increased slightly.When the site of GMPPA binding small molecule was mutated,the enzyme activity of the complex increased significantly.In the same way,after truncating the A Loop of GMPPA in the complex,the enzyme activity of the complex increased significantly,basically returning to the level of GMPPB monomer.Mutation impaired the interaction of the GMPPA/GMPPB complex.However,the overall enzyme activity of the complex improved,which further proved that GMPPA had a negative regulatory mechanism on the enzyme activity of GMPPB.GDP-mannose through E85 and Q247,which further negatively regulated the enzyme activity of GMPPB through A Loop(358-380).When the enzymatic reaction was in dynamic equilibrium,the concentrations of GDP-mannose in the GMPPA/GMPPB complex were lower than those in the GMPPB monomer.The GDP-mannose concentration of GMPPA E85K/Q247 A increased significantly,while the GDP-mannose concentration of GMPPB D218 A could not be detected.Small molecule binding sites played a key role in maintaining the equilibrium concentration of GDP-mannose.GMPPA and GMPPB played a synergistic regulatory role in maintaining the steady state of GDP-mannose.To further narrow down the key sites of A Loop,the 362P363N365 P of PPA in the complex was mutated to AAA.When the enzymatic reaction was in dynamic equilibrium,the GDP-mannose concentration of complex recovered to the GMPPB monomer level after the A Loop mutation,whereas the GDP-mannose concentration of complex also increased but failed to reach the GMPPB monomer level after the362P363N365 P mutation of PPA.A-Loop and its 362P363N365 P site played a key role in maintaining the equilibrium concentration of GDP-mannose.By comparing the enzyme activity of wild-type GMPPB and its G214 S and V111 G mutants,there was no difference between G214 S and wild-type GMPPB,while the activity of V111 G decreased significantly compared with wild-type GMPPB.The patient might be affected by GDP-mannose synthesis,then resulting in all kinds of diseases.The mutant plasmids of patients were successfully constructed by molecular cloning technology.The statistical results showed that the patient’s enzyme activity decreased to varying degrees.Conclusion:1.GMPPA mainly inhibited the enzyme activity of GMPPB,but GMPPA did not have enzyme activity.GMPPA showed the stronger ability of binding GDP-mannose than GMPPB.GMPPA may undergo conformational changes by binding GDP-mannose through E85 and Q247,which further negatively regulated the enzyme activity of GMPPB through A Loop(358-380).This whole process maintained the concentration of GDP-mannose at a reasonable level in the body through allosteric inhibition.The molecular mechanism of GMPPA/GMPPB complex regulating GDP-mannose homeostasis was clarified for the first time.2.The analysis of the complex structure revealed that the key sites for GMPPB binding GDP-mannose and enzyme activity were E80 and D218,which partially clarified the molecular mechanism.3.Compared with wild-type GMPPB,there was no significant change in the enzyme activity of G214 S,while the enzyme activity of V111 G and V111G/G214 S decreased significantly,which partially explained the pathogenic mechanism.