Gene Symbol: RNASE1

1. General

Pancreatic ribonuclease also known as ribonuclease A (RNase A) or ribonuclease 1 (RNase1) is an enzyme that catalyzes the breakdown of RNA and plays a role in the digestion of RNA in vertebrate species. Early work focused on bovine pancreatic RNase because of the large amount present in the pancreas. It has been described as the best studied enzyme of the 20^th century with four Nobel prizes awarded for studies of this protein (20). Owing to the high levels present in the bovine pancreas, RNase1 was historically considered as a digestive enzyme but with little purpose in humans and other non-ruminant mammals (2) where it exists at much lower concentrations. However, digestion of RNA occurs in the intestine of all species and RNase1 and other members of its superfamily are now known to have additional functions in host defense that will be discussed later.

Structure and Mechanism of Action

Bovine RNase1 was purified and crystalized by Kunitz in 1939 (16) and sequenced by Smyth, Stein and Moore in 1963 (30). It contains 124 amino acids, a calculated molecular weight of 13,683 and an excess of basic residues leading to an isoelectric point of 8.5 – 9.0. It contains four disulfide bonds and has been used as a model to study protein folding (22).  It is also glycosylated on asparagine residues with the glycosylated form originally termed RNase B. Because of the glycosylation and tertiary structure it runs on most SDS gels at 18 or 19 kDa. Porcine RNase 1 contains 125 amino acids (26); human RNase 1 contains 3 additional amino acids at the carboxyl terminal than bovine RNase1 (4). Bovine RNase1 was the first enzyme to have its three-dimensional structure determined. It has an overall shape like a kidney bean with the active site in the cleft containing His12, His119, and Lys41 (25). Other non-catalytic binding sites help the enzyme to form a complex with its polymeric substrate. RNase1 catalyzes the hydrolysis of 3’,5’-phosphodiester linkages in single stranded RNA when the base on the 3’ side is a pyrimidine (7, 11, 23). The process is usually represented as occurring in two stages with the first step involving the formation of a cyclic phosphodiester and the second it’s hydrolysis (23). RNase 1 does not hydrolyze DNA as it lacks a 2’-OH group. This allows it to be used to remove RNA contamination from DNA. It is also used in ribonuclease protection assays. RNase 1 forms dimers by domain swapping of amino termini by sulfhydryl bond formation in such a way as to keep each active site active (18). While retaining hydrolytic function the dimers acquire an additional biological activity with dimers, trimers and tetramers possessing anti-tumor activity

Ribonuclease A Superfamily

The human genome codes for 122 separate ribonucleases. The RNAse A superfamily consists of eight “canonical” ribonucleases with enzymatic activity and structural homology to RNase A (15). All are secretory proteins that share a disulfide bonded tertiary structure and are able to degrade RNA. All are coded for in a tight region of chromosome 14 in both humans and mice (27) and are believed to have originated from gene duplication (3, 31). Though the gene contains several exons, the coding region is contributed by a single exon. Understanding of their physiological role is incomplete but most are important for host defense and angiogenesis as well as digestion (9). The first member to be described is RNase 1 which in addition to being a pancreatic enzyme is produced in a variety of cells including vascular endothelial cells where after secretion it degrades vascular polymeric RNA and has anti-HIV-1 activity (15). RNase 2 and 3 are eosinophil secretory proteins termed eosinophil derived neurotoxin (EDN) and eosinophil cationic protein (ECP) respectively (15). RNase 4 is present in multiple tissues but it’s physiologic role is unclear. RNase 5, also known as angiogenin, induces blood vessel growth (10). RNase 7 is the most abundant RNase in skin while RNase 8 is expressed in the placenta (15). Additional genes in the cluster are related to ribonucleases (RNase 9 to 13) but their proteins have mutations preventing RNase activity. Some of the secreted RNases or their oligomers can enter cells and exert cytotoxic effects especially on tumor cells.

Ribonuclease Inhibitor

Mammalian ribonuclease inhibitor (RI) is a cytosolic protein of 50 KDa that binds with high affinity and 1:1 stoichiometry to pancreatic ribonuclease thereby inactivating it (8, 17). It is particularly abundant in placenta and liver and has been used to purify RNase.  It inhibits all members of the RNase A family. Its three-dimensional structure is that of a horseshoe which contains leucine rich repeats. Although the biological role is not clear it should bind and inactivate any RNase A family member escaping the secretory pathway and entering the cytoplasm.

2. Role of ribonuclease in the pancreas

Most cells contain millimolar concentrations of ribonucleotides but only micromolar concentrations of deoxyribonucleotides (5). Thus the diet contains a mixture of ribonucleoprotein, RNA and ribonucleotides. Nucleoproteins are broken down by pancreatic protease. The textbook view is that dietary nucleic acids are broken down by pancreatic RNase and DNase in the intestine. A recent study, however, has shown that pepsin in the stomach also hydrolyzes nucleic acid, so this digestion starts there (19). Foods rich in ribonucleoproteins include organ meats, seafood and legumes (5).

Pancreatic RNase (RNase 1) is present in all vertebrate pancreas but the amount varies greatly (2). Mammals with large amounts include ungulates, rodents and herbivorous marsupials. In the cow, RNase makes up 20% of digestive enzyme; this requirement is thought due to the large RNA load that is produced by bacteria from ruminal fermentation. In other species including humans, dog, cat and lower vertebrates, RNase is present at much lower amounts and may account for only 0.5 to 1% of pancreatic enzymes. Although only few studies exist, pancreatic RNase in all species appears to break down dietary nucleic acid in the gut lumen to nucleotides which are further broken down by intestinal alkaline phosphatase and 5’nucleotidase to nucleosides and free nitrogenous bases. Therefore, digestion of nucleic acids has a luminal and a brush border phase. These products are absorbed by enterocytes but most are excreted in urine; some are used for resynthesis primarily in the fasting state (13, 21, 29). Normally 80 to 90% of nucleotides are absorbed and these can become essential in certain diseases or periods of limited intake or rapid growth (5).

Ribonuclease is synthesized in acinar cells by rough ER, folded and packaged into zymogen granules beginning at 20 days gestation in the rat (33).  Folding occurs much more rapidly in cells compared to the isolated protein and except for a small amount of dimer any protein not ending as a folded monomer is degraded (12). It is secreted into the medium parallel to other digestive enzymes by pancreatic lobules and acini stimulated with CCK or cholinergic analogues (14, 24, 28). RNase 1 synthesis is reduced in experimental diabetes by 50% but much less than the decrease for amylase.

3. Tools for the study of Ribonuclease1

a. Antibodies

Biocompare (www.biocompare.com) lists 394 ribonuclease antibodies from 32 suppliers. Some are species specific while others are specific for RNase1 or other family members.  We have not tested any of these antibodies.

b. Ribonuclease activity

Early ribonuclease assays used the hydrolysis of yeast RNA; we used an assay described by Anfinsen (1) to measure the secretion of pancreatic ribonuclease by isolated rat pancreatic acini (24). These assays however are not specific for RNase1 and worked for the study of pancreatic acinar secretion because RNase1 is the major form present in pancreatic acini. An assay has also been described using the hydrolysis of cytidine 2’-3’-phosphate (6). Recently a quantitative fluorescence assay was developed based on the binding of ethidium bromide to yeast RNA (32).

4. References

1. Anfinsen CB, Redfield RR, Choate WL, Page J, and Carroll WR. Studies on the gross structure, cross-linkages, and terminal sequences in ribonuclease. J Biol Chem 207: 201-210, 1954. PMID: 13152095
2. Barnard EA. Biological function of pancreatic ribonuclease. Nature 221: 340-344, 1969. PMID: 4974403
3. Beintema JJ, and Kleineidam RG. The ribonuclease A superfamily: general discussion. Cell Mol Life Sci 54: 825-832, 1998. PMID: 9760991
4. Beintema JJ, Wietzes P, Weickmann JL, and Glitz DG. The amino acid sequence of human pancreatic ribonuclease. Anal Biochem 136: 48-64, 1984. PMID: 6201087
5. Carver JD, and Walker, WA. The role of nucleotides in human nutrition. J Nutr Biochem 6: 58 - 72, 1995.
6. Crook EM, Mathias AP, and Rabin BR. Spectrophotometric assay of bovine pancreatic ribonuclease by the use of cytidine 2':3'-phosphate. Biochem J 74: 234-238, 1960. PMID: 13812977
7. Cuchillo CM, Nogues MV, and Raines RT. Bovine pancreatic ribonuclease: fifty years of the first enzymatic reaction mechanism. Biochemistry 50: 7835-7841, 2011. PMID: 21838247
8. Dickson KA, Haigis MC, and Raines RT. Ribonuclease inhibitor: structure and function. Prog Nucleic Acid Res Mol Biol 80: 349-374, 2005. PMID: 16164979
9. Dyer KD, and Rosenberg HF. The RNase a superfamily: generation of diversity and innate host defense. Mol Divers 10: 585-597, 2006. PMID: 16969722
10. Fett JW, Strydom DJ, Lobb RR, Alderman EM, Bethune JL, Riordan JF, and Vallee BL. Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells. Biochemistry 24: 5480-5486, 1985. PMID: 4074709
11. Findlay D, Herries DG, Mathias AP, Rabin BR, and Ross CA. The active site and mechanism of action of bovine pancreatic ribonuclease. Nature 190: 781-784, 1961. PMID: 13699542
12. Geiger R, Gautschi M, Thor F, Hayer A, and Helenius A. Folding, quality control, and secretion of pancreatic ribonuclease in live cells. J Biol Chem 286: 5813-5822, 2011. PMID: 21156800
13. Ho CY, Miller KV, Savaiano DA, Crane RT, Ericson KA, and Clifford AJ. Absorption and metabolism of orally administered purines in fed and fasted rats. J Nutr 109: 1377-1382, 1979. PMID: 458492
14. Iwanij V, and Jamieson JD. Biochemical analysis of secretory proteins synthesized by normal rat pancreas and by pancreatic acinar tumor cells. J Cell Biol 95: 734-741, 1982. PMID: 6185502
15. Koczera P, Martin L, Marx G, and Schuerholz T. The Ribonuclease A Superfamily in Humans: Canonical RNases as the Buttress of Innate Immunity. Int J Mol Sci 17: 2016. PMID: 27527162
16. Kunitz M. Crystalline Ribonuclease. J Gen Physiol 24: 15-32, 1940. PMID: 19873197
17. Lee FS, and Vallee BL. Structure and action of mammalian ribonuclease (angiogenin) inhibitor. Prog Nucleic Acid Res Mol Biol 44: 1-30, 1993. PMID: 8434120
18. Libonati M. Biological actions of the oligomers of ribonuclease A. Cell Mol Life Sci 61: 2431-2436, 2004. PMID: 15526151
19. Liu Y, Zhang Y, Dong P, An R, Xue C, Ge Y, Wei L, and Liang X. Digestion of Nucleic Acids Starts in the Stomach. Sci Rep 5: 11936, 2015. PMID: 26168909
20. Marshall GR, Feng JA, and Kuster DJ. Back to the future: ribonuclease A. Biopolymers 90: 259-277, 2008. PMID: 17868092
21. McAllan AB. The degradation of nucleic acids in, and the removal of breakdown products from the small intestines of steers. Br J Nutr 44: 99-112, 1980. PMID: 6158999
22. Moore S, and Stein WH. Chemical structures of pancreatic ribonuclease and deoxyribonuclease. Science 180: 458-464, 1973. PMID: 4573392
23. Nogues MV, Vilanova M, and Cuchillo CM. Bovine pancreatic ribonuclease A as a model of an enzyme with multiple substrate binding sites. Biochim Biophys Acta 1253: 16-24, 1995. PMID: 7492594
24. Otsuki M, and Williams JA. Effect of diabetes mellitus on the regulation of enzyme secretion by isolated rat pancreatic acini. J Clin Invest 70: 148-156, 1982. PMID: 6177717
25. Raines RT. Ribonuclease A. Chem Rev 98: 1045-1066, 1998.
26. Reinhold VN, Dunne FT, Wriston JC, Schwarz M, Sarda L, and Hirs CH. The isolation of porcine ribonuclease, a glycoprotein, from pancreatic juice. J Biol Chem 243: 6482-6494, 1968. PMID: 5715511
27. Samuelson LC, Wiebauer K, Howard G, Schmid RM, Koeplin D, and Meisler MH. Isolation of the murine ribonuclease gene Rib-1: structure and tissue specific expression in pancreas and parotid gland. Nucleic Acids Res 19: 6935-6941, 1991. PMID: 1840677
28. Scheele GA, and Palade GE. Studies on the guinea pig pancreas. Parallel discharge of exocrine enzyme activities. J Biol Chem 250: 2660-2670, 1975. PMID: 1123325
29. Schwenk M, Hegazy E, and Lopez del Pino V. Uridine uptake by isolated intestinal epithelial cells of guinea pig. Biochim Biophys Acta 805: 370-374, 1984. PMID: 6210111
30. Smyth DG, Stein WH, and Moore S. The sequence of amino acid residues in bovine pancreatic ribonuclease: revisions and confirmations. J Biol Chem 238: 227-234, 1963. PMID: 13989651
31. Sorrentino S. The eight human "canonical" ribonucleases: molecular diversity, catalytic properties, and special biological actions of the enzyme proteins. FEBS Lett 584: 2194-2200, 2010. PMID: 20388512
32. Tripathy DR, Dinda AK, and Dasgupta S. A simple assay for the ribonuclease activity of ribonucleases in the presence of ethidium bromide. Anal Biochem 437: 126-129, 2013. PMID: 23499964
33. Van Nest GA, MacDonald RJ, Raman RK, and Rutter WJ. Proteins synthesized and secreted during rat pancreatic development. J Cell Biol 86: 784-794, 1980. PMID: 7410479