Key achievements

In his Ph.D. work S.O. studied proteins interacting with pre-mRNA and mature mRNA and demonstrated that the proteins are partly different.

Since 1971 S.O. has studied the mechanism of action of certain protein toxins made by plants and pathogenic bacteria. These toxins comprise diphtheria toxin, Shigella toxin, ricin, abrin, modeccin and others, and they are some of the most potent poisons known. This property is the reason for their current use to form immunotoxins, where monoclonal antibodies to cell surface markers are armed with toxins for targeted cell killing in cancer and other diseases. Other important applications of the toxins is their use as probes in studies on endocytosis and intracellular vesicular transport and on translocation of proteins across membranes.

Common for these toxins is that they consist of two functionally different parts, one (the B-moiety) that binds to cell surface receptors and one (the A-moiety) that has enzymic activity and is able to inactivate in a specific manner ribosomes or elongation factor 2 and thereby inhibit protein synthesis. Since the target is in the cytosol, the toxins, or their enzymatically active part must cross the cell membrane to act.

He has studied the mechanism of intracellular pH-regulation and more recently the ability of acidic fibroblast growth factor to penetrate across cellular membranes into the cytosol and the nucleus.

Main scientific contributions:

The main contributions of S.O. and collaborators are the following (some key references are given in parenthesis):

• Discovery that the plant toxins abrin and ricin inhibit cell-free protein synthesis and that the toxins inactivate ribosomes catalytically. (Olsnes and Pihl, FEBS Letters, 20 (1972) 327-329, FEBS Letters, 28 (1972) 48-50 and Nature, 238 (1972) 459-461).
• Discovery of the A-chain/B-chain structure of abrin and ricin. Demonstration that the A-chain inhibits protein synthesis, whereas the B-chain binds to cell surface receptors containing terminal galactose. Olsnes and Pihl, Eur.J.Biochem., 35 (1973) 179-185 and Biochemistry, 12 (1973) 3121-3126; Olsnes et al. J.Biol.Chem.249 (1974) 803-810; Refsnes et al. J.Biol.Chem. 249 (1974) 3557-3562; Sandvig et al. J.Biol.Chem. 251, (1976) 3977-3984; Olsnes et al. J.Biol.Chem. 251, (1976) 3985-3992).
• Formation of the first hybrid toxins (in a reciprocal manner between the A- and the B-chains of abrin and ricin). (Olsnes Nature, 249 (1974) 627-631 and J.Immunol. 113 (1974) 842-847).
• First isolation and characterization of modeccin, a plant toxin related to abrin and ricin. Refsnes et al. Biochem.Biophys. Res.Commun. 79 (1977) 1176-1183; Olsnes et al.J.Biol.Chem. 253 (1978) 5069-5073).
• First isolation of Shigella toxin and elucidation of its subunit structure. Demonstration that the A1-fragment inhibits protein synthesis by inactivating catalytically the 60 S ribosomal subunit. (Olsnes and Eiklid, J.Biol.Chem. 255, (1980) 284-289; Olsnes et al. J.Biol.Chem. 256, (1981) 8732-8738; Reisbig et al.J.Biol.Chem. 256, (1981) 8781-8744).
• Demonstration that the mistletoe-toxin, viscumin, resembles ricin in structure and function. (Olsnes et al. J.Biol.Chem. 257 (1982) 13263-13270; Stirpe et al. J.Biol.Chem. 257 (1982) 13271-13277).
• Discovery that low pH is required for entry of diphtheria toxin to the cytosol, implicating endocytic uptake. Development of a system for direct translocation of the toxin through the plasma membrane. (Sandvig and Olsnes, J.Cell Biol. 87, (1980) 828-832 and J. Biol. Chem. 256, (1981) 9068-9076; Moskaug et al.J.Biol.Chem.262 (1987) 10339-10345 and J.Biol.Chem. 263 (1988)2518-1525).
• Demonstration that endocytosis is involved in the entry of abrin, ricin, modeccin, viscumin and shigella toxin and that transport to the trans-Golgi network is essential for intoxication. (Sandvig and Olsnes, J.Biol.Chem. 257, (1982) 7495-7503 and J.Biol.Chem. 257, (1982) 7504-7513; Sandvig et al. J.Cell Biol. 98 (1984), 963-970 and Cancer Res. 46(1986) 6418-6422; van Deurs et al.J.Cell.Biol. 102 (1986) 37-47).

• Discovery of the requirement of a trans-membrane H+-gradient and of permeant anions for translocation of diphtheria toxin to the cytosol. (Sandvig and Olsnes, J.Cell Physiol. 119 (1984) 7-14 and J.Biol.Chem. 261 (1986) 1570-1575; Sandvig et al.J.Biol.Chem.261 (1986) 11639-11644).
• Evidence for an alternative route of endocytosis. Demonstration that acidification of the cytosol blocks endocytosis of ligands from coated pits, but not the uptake and action of ricin. (Sandvig et al.J.Cell.Biol. 105 (1987) 679-689 and J.Cell. Biochem. 36 (1988) 73-81).
• Discovery of regulation by intracellular pH of Na+-independent chloride/bicarbonate antiport. Demonstration that Na+-dependent and Na+-independent Cl-/HCO3- antiport play opposite roles in pH-homeostasis. (Olsnes et al. J.Cell Biol. 102 (1986) 967-971, Biochemistry, 26 (1987) 2778-2785 and J.Cell. Physiol. 132 (1987) 192-202; Tønnessen et al.J. Cell. Physiol.132 (1987) 183-191; Madshus and Olsnes, J.Biol.Chem.262 (1987) 7486-7491).
• Discovery that diphtheria toxin and anthrax tocin can be used as a carrier to translocate peptides and proteins into the cytosol. (Stenmark et al. J. Cell Biol. 113 (1991) 1025-1032; Wiedlocha et al., Embo J. 11 (1992) 4835-4842; Wesche et al., Biochemistry 37 (1998) 213-346).
• First demonstration that diphtheria toxin A-fragment requires unfolding before translocation to the cytosol can take place. (Falnes et al., J. Biol. Chem. 269 (1994) 8402-8407; J. Biol. Chem.270 (1995) 20787-20793).
• Evidence for two modes of signal transduction by acidic fibroblast growth factor and for the ability of the growth factor to enter into cells. Observation that growth factor translocated to the cytosol as a fusion protein with diphtheria toxin is translocated to the nucleus and induces DNA synthesis in cells lacking growth factor receptors. (Wiedlocha et al. Cell, 76 (1994) 1039-1051; J. Biol. Chem. 270 (1995) 30680-30685; Mol. Cell. Biol. 16 (1996) 270-280; Klingenberg et al. J. Biol. Chem. 273 (1998) 11164-11172; 276 (1999) 11972-11980; J. Cell Sci. 113 (2000) 1827 1838).
• Cloning and characterization of a novel nuclear protein, FIBP, that binds mitogenic acidic fibroblast growth factor. (Kolpakova et al. Biochem. J. 336 (1998) 213-222).
• First demonstration that ricin is transported retrograde from the Golgi apparatus to the endoplasmic reticulum and translocated to the cytosol by the sec61 complex. (Rapak et al. Proc. Natl. Acad. Sci. USA 94 (1997) 3783-3788; Wesche et al. J. Biol. Chem. 274 (1999) 34443-34449).

Significance of the work

Toxins with a structure-function relationship as in those S.O. and collaborators have studied represent main pathogenicity factors of a large group of infectious diseases. Thus, diphtheria, cholera, pertussis, tetanus and botulism are diseases where the symptoms are due to toxins with an A-B structure and where the intracellular action is enzymic. In Pseudomonas infections and in dysentery the role of the toxin is less dominating, but in both cases the toxins play a role in tissue damage. Shiga-like toxin is produced by an number of enteropathogenic E.coli strains, and it is the causative agent in hemolytic uremic syndrome (the “hamburger disease”) which has become a serious problem. Toxins produced in antrax and in certain Clostridia also have a structure-function relationship similar to the A-B toxins.

Since S.O. and collaborators constructed the first hybrid toxins in 1974, a large number of laboratories have prepared hybrids of ricin or diphtheria toxin with antibodies against cell surface markers, particularly against tumor markers, in attempts to construct specific toxins for targeted cell killing. Such conjugates are now being tried in many cancer centers and in bone marrow transplantation units. A problem with these constructs is, however, that they are often not very efficient. It is therefore a common feeling that more knowledge about the entry mechanism of the native toxins is required in order to be able to tailor correctly efficient immunotoxins.

The fact that toxins of the A-B type have a complicated mechanism of action make them interesting tools to explore normal cellular mechanisms. Over the years S.O. and collaborators have used the toxins in model systems to study ligand-receptor interactions, endocytosis, intracellular traffic between different vesicular compartments, transport of endocytosed material back to the cell surface and protein translocation. The toxin is transported retrograde through the Golgi complex to the endoplasmic reticulum where it is translocated across the membrane to the cytosol. This is probably the entry route for many toxins including most immunotoxins.

The toxins are the only known group of proteins that are translocated across membranes in the direction from the exterior to the cytosol, but resent data from S.O. and collaborators indicate that also physiological proteins such as acidic fibroblast growth factor are translocated in a similar way. The mechanism of toxin translocation has properties in common with translocation of export proteins across cellular membranes. The ability of toxins to carry peptides and proteins into the cytosol has later been exploited by other groups in vaccine development (MHC-class I immunity) with promising results.

Work on anion transport and pH-regulation is important for several reasons. Many cellular processes, including growth and division are regulated by the intracellular pH. Furthermore, anion transport plays an important role in transport of salt across membranes, a process that is deficient or disturbed in many diseases.

The finding that acidic fibroblast growth factor translocated to the cytosol as a fusion protein with diphtheria toxin enters the nucleus and stimulates DNA synthesis in cells lacking FGF-receptors indicates that the growth factor does not only act by stimulating the tyrosine kinase of the FGF-receptor. Later work indicates that also the growth factor as such has the capability of entering the cell, possibly by a mechanism employed by certain toxins.