Identification of PDI family members required for the productionof useful proteins
Hiroshi KADOKURA
Institute of Multidisciplinary Research for Advanced Materials,Tohoku University
Research aims
A disulfide bond is a covalent linkage formed by two cysteines on a protein. Formation ofdisulfide bonds is catalyzed in vivo and is a critical step in the folding of a great number ofsecretory and membrane proteins1). Many of commercially valuable mammalian proteinssuch as human growth hormones are secretory proteins with disulfide bonds. A major
obstacle for the production of recombinant secretory proteins of mammalian origin in
microorganisms is the difficulty in forming correct disulfide bonds. This problem is probably
caused by differences in the protein disulfide bond formation systems of microorganisms andmammalian cells. In the endoplasmic reticulum (ER) of mammalian cells, for instance, there
are ~20 protein disulfide isomerase (PDI) family members that are thought to catalyze theformation of disulfide bonds in secretory and membrane proteins. The high number ofdifferent enzymes probably reflects the need to introduce disulfide bonds into a great varietyof secretory proteins present in mammalian cells. However, the physiological roles of manyof the PDI family members remain unclear. Insulin, a peptide hormone produced bypancreatic β-cells, plays an important role in the maintenance of blood glucose levels.
Insulin has three disulfide bonds. For the folding and activity of insulin, it is essential that all
three disulfide bonds are correctly formed. However, the enzyme(s) responsible for thisprocess remain unclear. To identify PDI family members that are required for insulin
production, we explored the physiological substrates of PDIp, a PDI family member that is
specifically expressed in the pancreas, and identified the PDI family members that directlyassociate with proinsulin.
Methods
During catalysis of disulfide bond formation, PDI family members form a disulfide-linked
complex with their substrate1). We have established methods to stabilize such
enzyme-substrate complexes that arise in cultured cells or in animal tissue by treating
samples with 10% trichloroacetic acid and 50 mM N-ethylmaleimide2). After theenzyme-substrate complexes were purified using an antibody, they were subjected to mass
spectroscopic analysis for the identification of either the substrates of PDIp or PDI familymember(s) that directly interact(s) with proinsulin.
Results
Identification of the substrates of PDIp, a PDI family member specifically expressed in thepancreas
PDIp is a PDI family member specifically expressed in the pancreas, which produces
hormones such as insulin and digestive enzymes such as trypsin. However, the physiological
functions of PDIp remained unclear. Here, we aimed to identify the endogenous substrates ofthis enzyme in order to understand its function. In particular, we wished to know whether or
not PDIp plays a role in the production of insulin. By combining acid quenching and thiol
alkylation, we stabilized and purified the complexes formed between PDIp and its substrate
proteins from mouse pancreas. Further mass spectrometry analysis of the complexes enabled
us to identify the disulfide-linked partners of PDIp in vivo and find that PDIp interacts
directly with pancreatic digestive enzymes but not with pancreatic hormones such as insulinand glucagon. Pancreas consists mainly of endocrine cells that produce hormones such asinsulin and exocrine cells that produce digestive enzymes. We separated mouse pancreatic
endocrine and exocrine cells and studied the distribution of PDIp using an anti-PDIp
antibody to find that PDIp is expressed in the exocrine cells but not in the endocrine cells,
thereby excluding the possibility that PDIp may play a role in the production of insulin.
Interestingly, when one of the identified proteins was expressed in cultured cells, its
proenzyme formed disulfide-linked aggregates within cells. However, co-expression of PDIpwith the protein prevented the formation of aggregates and enhanced the secretion ofproenzyme into the medium. Strikingly, we failed to see such effect when we co-expressed
other PDI family members with the protein. Thus, PDIp is likely a PDI family member thathas evolved to assist in the biosynthesis of digestive enzymes3). These findings should
provide important information for the analysis of the causes of diseases related to thepancreas and for the establishment of an efficient system for the production of digestive
enzymes of human origin in microorganisms.
Identification of PDI family members that directly interact with proinsulin
Disulfide bond formation in insulin takes place while insulin exists as proinsulin in the ERof pancreatic β-cells. Upon folding, proinsulin is transported to secretory vesicles andprocessed to form mature insulin. As we have already described above, during catalysis ofdisulfide bond formation, PDI family members form disulfide-linked complexes with theirsubstrates. We adopted this property to identify the enzymes involved in insulin disulfide
bond formation. By treating an insulin-producing pancreatic β-cell line, MIN6, withtrichloroacetic acid and N-ethylmaleimide, we stabilized the disulfide-linked complexesbetween (a) PDI family member(s) and proinsulin4). We then purified the complexes using
an antibody against proinsulin and analyzed them by mass spectrometry. This analysis led tothe discovery of four PDI family members that directly interacted with proinsulin via (an)intermolecular disulfide bond(s). Interestingly, the phenotype obtained by knocking down
one of these proteins in MIN6 was different from that obtained by knocking down anotherprotein, suggesting that the two enzymes acted at different steps in the folding of proinsulin.
Conclusion
In this study, we discovered that PDIp is required for the biosynthesis of digestive
enzyme(s), but not for that of insulin. Although PDIp is evolutionarily conserved amongvertebrates, it remained unclear why this enzyme is expressed specifically in the pancreas.
We have elucidated this with our current study3). Furthermore, these findings should
aid the establishment of an efficient system for the production of digestive
enzymes of human origin in microorganisms.
We have also successfully identified four PDI family members that interact withproinsulin via an intermolecular disulfide bond(s) using a pancreatic β–cell line. Interestingly,
the phenotype obtained by knocking down one of these proteins in the cell was different
from that obtained by knocking down another protein, suggesting that the two enzymes acted
at different steps in the biosynthesis of insulin. The findings obtained in this study provide animportant basis for future studies.
References1) Kadokura, H., Katzen, F., Beckwith, J. (2003) Protein disulfide bond formation inprokaryotes. Ann. Rev. Biochem. 72: 111-135.
2) Fujimoto, T., Inaba, K., and Kadokura, H. (2019) Methods to identify the substrates ofthiol-disulfide oxidoreductases. Protein Sci. 28: 30-40.
3) Fujimoto, T. et al. (2018) Identification of the physiological substrates of PDIp, apancreas-specific protein disulfide isomerase family member. J. Biol. Chem. 293:
18421-18433.
4) Tsuchiya Y. et al. (2018) IRE1-XBP1 pathway regulates oxidative proinsulin folding inpancreatic β cells. J. Cell Biol. 217: 1287-1301.