Expression Of Recombinant Secretable FNDC5 In Pichia Pastoris And Detection Of Its Biological Activity

ObjectiveObesity,which is associated with the development of various metabolic diseases,has been highlighted as a priority public health problem worldwide in recent decades.Irisin,an exercisedriven hormone,was first identified in 2012 and was presumably cleaved from its precursor protein fibronectin type Ⅲ domain containing 5(FNDC5).The main function of irisin is to induce the "browning" of white adipocytes by increasing UCP-1 and consequently increasing whole-body energy expenditure.Therefore,irisin has attracted much attention in the treatment of obesity and related metabolic diseases.Increasing evidence has suggested that FNDC5 may have more than one type of transcript.In our previous study,a novel secretable FNDC5(sFNDC5)isoform lacking the transmembrane region was found in rat INS-1 cells and multiple rat tissues.Considering that sFNDC5’s major distinction from irisin is that it lacks the transmembrane domain while the majority of irisin sequences are shared,a range of similar functions,such as browning and lipolysis,and even its biological functions compared with irisin,need to be further explored.To explore the function of sFNDC5,we chose the methylotrophic yeast Pichia pastoris as an efficient tool for the large-scale production of high purity recombinant secretable FNDC5(r-sFNDC5).The biological activities of r-sFNDC5 in energy expenditure,browning,and lipolysis were further explored and compared with those of irisin in adipocytes.Methods1.Expression plasmid construction and transformation of P.pastoris(pPICZaA-sFNDC5 plasmid).2.Expression and purification of r-sFNDC5.3.To confirm glycosylation of the r-sFNDC5 protein,we treated the protein with recombinant N-glycanase and analyzed it by SDS-PAGE.4.Differentiation of 3T3-L1 preadipocytes into mature adipocytes5.Cell proliferation detection was used the CCK-8 assay.6.Western blot was used to measure the protein expression of UCP1,HSL,perilipin,and other lipid metabolism related protein in 3T3 L1 cells after r-sFNDC5 and r-irisin treatment.7.RNA isolation from 3T3 L1 cells after r-sFNDC5 and r-irisin treatment.RT-qPCR to measure the expression of related mRNA.8.Immunofluorescence(IF)staining was used to detect the expression of UCP-1.9.Adipogenic differentiation was confirmed by Oil Red O staining.10.Intracellular ATP detection was used to detect cellular energy metabolism.11.All data are presented as the means ± SEM of at least three independent experiments.The statistical significance was analyzed by using GraphPad Prism 7.0 software,and comparisons between two groups were performed using one-way ANOVA followed by unpaired Student’s t-test.P<0.05 was considered statistically significant.Results1.Sequence analysis of sFNDC5Through alignment of the amino acid sequences of this new sFNDC5 transcript and membrane bound FNDC5(mFNDC5),we found that this variant lacks a transmembrane domain(exon 5)but shares most of the irisin sequence.2.Expression and Purification of r-sFNDC5 in P.pastorisWe obtained the sFNDC5 protein with a P.pastoris yeast expression system and purified it with a Ni-NTA column.SDS-PAGE analysis showed purified r-sFNDC5 with a molecular weight range from 18 to 27 kDa.3.N-linked glycosylation analysis of r-sFNDC5The enzyme-treated r-sFNDC5 exhibited a single band with the expected molecular mass of 18 kDa,confirming that the 20-27 kDa mass of r-sFNDC5 expressed by P.pastoris was mainly the result of N-glycosylation.4.r-sFNDC5 stimulates browning and lipolysis in differentiated mature 3T3-L1 cells(1)CCK8 assay showed that no effect on cell viability at 20,50 and 100 nM concentrations of r-sFNDC5 treatment,which indicated no toxicity of this protein.(2)r-sFNDC5 induced a rapid upregulation of browning(UCP-1,PRDM16,Cidea)and lipolysis-related genes(ATGL,HSL)in differentiated mature 3T3-L1 adipocytes after treatment for 8 h.(3)UCP-1 and ATGL protein levels were also significantly enhanced after r-sFNDC5 treated.(4)Immunofluorescence staining of UCP-1 further confirmed a significantly higher level of expression after treatment with r-sFNDC5.(5)Intracellular ATP levels were decreased with r-sFNDC5 treatment.5.r-sFNDC5 inhibits adipogenic differentiation of 3T3-L1 cells(1)The 3T3-L1 cells were treated with different concentrations(20 nM and 50 nM)of rsFNDC5 throughout the differentiation period.Oil Red O staining showed adipocyte accumulation was reduced in the presence of r-sFNDC5 after 10 days of differentiation.(2)The expression of Perilipin and Adipoq was also reduced at both the gene and protein levels.6.Functional comparison of r-sFNDC5 and r-irisin in differentiated mature 3T3-L1 adipocytesDifferentiated mature 3T3-L1 adipocytes with 20 nM r-sFNDC5 and r-irisin for 8 h and found that both proteins induced the expression of genes and proteins related to lipid metabolism,r-sFNDC5 had a much stronger effect than r-irisin.Conclusion1.We first obtained the sFNDC5 protein with a P.pastoris yeast expression system and purified it with a Ni-NTA column,which is a useful experimental tool for heterogeneous protein production.2.r-sFNDC5 is a N-linked glycosylated protein.3.r-sFNDC5 increased browning,lipolysis,and mitochondrial biogenesis genes at both the transcriptional and protein levels.4.r-sFNDC5 exerts an inhibitory effect on preadipocyte adipogenic differentiation.5.r-sFNDC5 was proven superior to r-irisin in terms of functions related to lipid metabolism.