Novozymes (United Kingdom)

Albumin is the most abundant protein in blood where it has a pivotal role as a transporter of fatty acids and drugs. Like IgG, albumin has long serum half-life, protected from degradation by pH-dependent recycling mediated by interaction with the neonatal Fc receptor, FcRn. Although the FcRn interaction with IgG is well characterized at the atomic level, its interaction with albumin is not. Here we present structure-based modelling of the FcRn–albumin complex, supported by binding analysis of site-specific mutants, providing mechanistic evidence for the presence of pH-sensitive ionic networks at the interaction interface. These networks involve conserved histidines in both FcRn and albumin domain III. Histidines also contribute to intramolecular interactions that stabilize the otherwise flexible loops at both the interacting surfaces. Molecular details of the FcRn–albumin complex may guide the development of novel albumin variants with altered serum half-life as carriers of drugs. Albumin transport proteins circulate in the blood and are protected from degradation by interaction with the neonatal Fc receptor. Andersenet al. investigate the albumin binding site of the neonatal Fc receptor and find pH sensitive ionic networks at the binding interface.

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals. A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.

Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major Zn2+ site on albumin is a 5-coordinate site with average Zn-O/N distances of 1.98 Å and a weak sixth O/N bond of 2.48 Å, consistent with coordination to His67 and Asn99 from domain I, His247 and Asp249 from domain II (residues conserved in all sequenced mammalian albumins), plus a water ligand. The dynamics of the domain I/II interface, thought to be important to biological function, are affected by Zn2+ binding, which induces cooperative allosteric effects related to those of the pH-dependent neutral-to-base transition. N99D and N99H mutations enhance Zn2+ binding but alter protein stability, whereas mutation of His67 to alanine removes an interdomain H-bond and weakens Zn2+ binding. Both wild-type and mutant albumins promote the safe management of high micromolar zinc concentrations for cells in cultures. Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major Zn2+ site on albumin is a 5-coordinate site with average Zn-O/N distances of 1.98 Å and a weak sixth O/N bond of 2.48 Å, consistent with coordination to His67 and Asn99 from domain I, His247 and Asp249 from domain II (residues conserved in all sequenced mammalian albumins), plus a water ligand. The dynamics of the domain I/II interface, thought to be important to biological function, are affected by Zn2+ binding, which induces cooperative allosteric effects related to those of the pH-dependent neutral-to-base transition. N99D and N99H mutations enhance Zn2+ binding but alter protein stability, whereas mutation of His67 to alanine removes an interdomain H-bond and weakens Zn2+ binding. Both wild-type and mutant albumins promote the safe management of high micromolar zinc concentrations for cells in cultures. Zinc is not only required for hundreds of essential extra- and intracellular proteins and enzymes but is also recruited by toxins such as anthrax lethal factor (1Turk B.E. Wong T.Y. Schwarzenbacher R. Jarrell E.T. Leppla S.H. Collier R.J. Liddington R.C. Cantley L.C. Nat. Struct. Mol. Biol. 2004; 11: 60-66Crossref PubMed Scopus (176) Google Scholar) and staphylococcal enterotoxin (2Petersson K. Håkansson M. Nilsson H. Forsberg G. Svensson L.A. Liljas A. Walse B. EMBO J. 2001; 20: 3306-3312Crossref PubMed Scopus (86) Google Scholar). There is a need to understand how zinc transport and distribution is controlled (3Ford D. Proc. Nutr. Soc. 2004; 63: 21-29Crossref PubMed Scopus (48) Google Scholar). Although considerable progress has been made in the identification and study of membrane-bound zinc transporters, the molecular mechanism of extracellular zinc transport is still obscure. The total concentration of zinc in blood is high, ∼15–20 μm (4Caroli S. Alimonti A. Coni E. Petrucci F. Senofonte O. Violante N. Crit. Rev. Anal. Chem. 1994; 24: 363-398Crossref Scopus (259) Google Scholar), and plasma zinc concentrations are maintained at a relatively constant level, except during periods of dietary zinc depletion and acute responses to stress or inflammation, when they are depressed (5Cousins R.J. Clin. Phys. Biochem. 1986; 4: 20-30PubMed Google Scholar). In humans, ∼98% of so-called “exchangeable” zinc in blood plasma (9–14 μm) is bound to serum albumin (6Giroux E.L. Henkin R.I. Bioinorg. Chem. 1973; 2: 125-133Crossref Scopus (42) Google Scholar). Studies on perfused rat intestine have implicated albumin in the transport of newly absorbed zinc in portal blood, from the intestine to the liver (5Cousins R.J. Clin. Phys. Biochem. 1986; 4: 20-30PubMed Google Scholar). Albumin has also been shown to promote zinc uptake by endothelial cells, with receptor-mediated endocytosis as the most likely mechanism (7Rowe D.J. Bobilya D.J. Proc. Soc. Exp. Biol. Med. 2000; 224: 178-186Crossref PubMed Scopus (37) Google Scholar). Albumin, the most abundant protein in blood plasma (∼40 mg ml−1 and 0.6 mm), is synthesized in the liver and secreted into the blood stream as a 585-residue, single-chain protein after loss of a 24-residue propeptide (8Peters Jr., T. All about Albumin: Biochemistry, Genetics and Medical Applications. Academic Press, New York1996: 9-54Google Scholar). The protein is largely α-helical and folds into three structurally homologous domains (I, II, and III), each of which contains two subdomains (A and B) (see Fig. 1A) (9Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1523) Google Scholar). There are 35 Cys residues that form six disulfide bridges in each domain, except for domain I, which contains only five bridges and a free thiol at Cys34. Zinc binding to albumin has also been demonstrated in vitro, and the so-called high-affinity site for zinc on albumin displays a binding constant of K ≈ 107m−1 (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 11Goumakos W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, 12Ohyoshi E. Hamada Y. Nakata K. Kohata S. J. Inorg. Biochem. 1999; 75: 213-218Crossref PubMed Scopus (79) Google Scholar, 13Bal W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar). Although over 50 x-ray structures of albumin have been reported to date (9Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1523) Google Scholar, 14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1190) Google Scholar, 15Carter D.C. He X.M. Munson S.H. Twigg P.D. Gernert K.M. Broom M.B. Miller T.Y. Science. 1989; 244: 1195-1198Crossref PubMed Scopus (536) Google Scholar, 16Ghuman J. Zunszain P.A. Petitpas I. Bhattacharya A.A. Otagiri M. Curry S. J. Mol. Biol. 2005; 353: 38-52Crossref PubMed Scopus (1488) Google Scholar), no experimental structural data are available for the zinc site on albumin. On the basis of 111Cd NMR studies, site-directed mutagenesis, and molecular modeling, we have proposed that the major zinc site on albumin, termed site A, is located at the interface of domains I and II and is formed by the side chains of His67 and Asn99 from domain I and by His247 and Asp249 from domain II (Fig. 1B) (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, 18Lu J. Stewart A.J. Sadler P.J. Pinheiro T.J. Blindauer C.A. Biochem. Soc. Trans. 2008; 36: 1317-1321Crossref PubMed Scopus (194) Google Scholar). Here, we report the first direct structural characterization of a zinc site on albumin, using Zn K-edge x-ray absorption fine structure (EXAFS) 2The abbreviations used are: EXAFSx-ray absorption fine structureN-to-Bneutral-to-base transitionrHArecombinant human albumin. spectroscopy. We also explored the possibility of engineering albumins with increased or decreased zinc-binding affinity by mutating the postulated zinc-binding ligands. NMR methods were used to identify mutated and zinc-binding histidines and to probe the effects of zinc binding on the conformational dynamics of the protein. Finally, we show that albumin at physiological concentrations promotes the culture of hepatocytes at otherwise toxic zinc concentrations. x-ray absorption fine structure neutral-to-base transition recombinant human albumin. All albumin samples were prepared from purified, recombinant proteins produced as described previously (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar). albumin were prepared from a human albumin by site-directed were by of samples with and NMR were at K on a at using a and probe using albumin in at to the for a in using for be to by 50 50 and as to were for the and domain data using a for water data are shown and described in 111Cd NMR were on of mutant albumins in 50 50 and of prepared by in the of NMR were at K using a probe with as an Albumin samples were in 50 which been using to albumin of and were from Zn K-edge data were on and at at K and in data were and The data were and as described previously M. I. W. D.J. Sadler P.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) using We used of the zinc site (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar), on a structure at Å of (9Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1523) Google as a for data The the side chains of and as as the side and of His247 and all of the bond to Asp249 required of the In to data and the molecular modeling using an of the The of as by J. Google Scholar) and G. PubMed Scopus Google Scholar). The of the were by to the wild-type (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar), using a were using R. M. K. J. Mol. PubMed Scopus Google Scholar) and The affinity of albumin for zinc using an C.A. 2004; PubMed Scopus Google Scholar). of μm recombinant human albumin for at K of and The free zinc concentration by using concentrations of The free zinc concentrations were using the of the G. and Academic Scholar), and the for the J. Chem. Soc. Scopus Google Scholar). the samples were to to or the zinc zinc that bound to albumin the The albumin concentration by the at using a of J. NMR Studies of of Scholar). The zinc concentration using the zinc μm Zn2+ in were in to albumin The data are the are on in albumin cells were from the and maintained in culture in with serum and and which the zinc-binding and at a concentration of were at in an and with or as cells they were using and were culture at using of (see Fig. or of zinc Fig. and to The and the with were with of with or μm in the or of wild-type or mutant albumin with as a a the were using to samples to to were and using a The total of after to for All were in on in as the residues in high-affinity zinc binding on albumin, we the of wild-type and the three and N99H by Zn K-edge and 111Cd and NMR spectroscopy. The mutant to a of and to zinc and in a wild-type and the two Asn99 were to the weak with the or the wild-type we Zn K-edge data for three and (see and Fig. is that albumin contains (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 13Bal W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar), important to that the methods zinc binding to high-affinity of on previously data (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 11Goumakos W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, 13Bal W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar, T. Inorg. Scopus Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar), the is to albumin with bound to the Sarkar B. Chem. Scopus Google Scholar), bound to the so-called site P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar), and Zn2+ to site (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar). The for albumins are that the the first of Zn2+ to site A. and for the mutant proteins were on of Zn2+ and albumin. The data for the and are with that of wild-type in Fig. The data show that each mutation the zinc of the data a structural of the zinc site in wild-type albumin. of the first or for wild-type of bound Zn2+ the zinc as a 5-coordinate O/N site with an average Zn-O/N of 1.98 Å, plus O/N at 2.48 molecular on Protein in which zinc is bound by the postulated and a water The data for an of with to the experimental to as the and data (Fig. with of the of the a of the site (Fig. The of His67 the side of Asn99 of His247 and of Asp249 with a water the first whereas the of His247 a bound to the data by by the or of Asp249 or Asn99 as the sixth not and residues are in to all of residues Å of the Zn2+ in to for all to the not be from the data for a of the and a of related with structures were the of the of residues shown in Fig. are of a of which a to the data The of the mutant (Fig. from the of wild-type and is consistent with a The loss of is also in the of the first in the In for the Asn99 form the (Fig. of wild-type All data for the were using on the in mutation of the residues Fig. The affinity of the mutated albumins for zinc by The high zinc affinity of albumins reported in the K (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 11Goumakos W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google E. Hamada Y. Nakata K. Kohata S. J. Inorg. Biochem. 1999; 75: 213-218Crossref PubMed Scopus (79) Google W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar) be to the of the postulated site and the of the side of as of the a binding that is relatively weak and in of Asn99 to or of which have high for to zinc whereas the of the His67 to Fig. a of zinc from the of K be for the binding constant of the first of Zn2+ to wild-type at a in the as those reported previously for human serum albumin (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 11Goumakos W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, 12Ohyoshi E. Hamada Y. Nakata K. Kohata S. J. Inorg. Biochem. 1999; 75: 213-218Crossref PubMed Scopus (79) Google Scholar, 13Bal W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar). all three mutations have a on the affinity of albumin for The the N99H The binding of the Asn99 are that of wild-type whereas that of the mutant is data are in with the on side chains with affinity and on the and affinity on the into the postulated zinc binding site G. and Academic Scholar). 111Cd and NMR spectroscopy were used to probe the binding of and Zn2+ to the mutant albumins in to wild-type 111Cd and NMR have been used previously to probe zinc in proteins albumin W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, T. Inorg. Scopus Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar). with previously reported data W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, T. Inorg. Scopus Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar), wild-type albumin with 111Cd to two in 111Cd NMR at and B) (Fig. The 111Cd NMR of as that for wild-type at and (Fig. that site is affected by the Asn99 whereas site The of Zn2+ and are also of Zn2+ were not to from site Fig. in to wild-type for which of after of of Zn2+ (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar). affinity for with Zn2+ on of an by the in the N99H mutant is in with the J. Chem. Soc. Scopus Google Scholar). The 111Cd NMR of at K only which is by the in of the affinity of mutant for Zn2+ and affinity for of of and the to two at and (Fig. The at be to a by with a Fig. whereas the at is in the for a site with and three to five Biochem. Cell Biol. 1998; PubMed Google Scholar). The of the that in the N99D which are at an on the NMR at K. We used NMR spectroscopy to identify residues of and to probe the of Zn2+ on residues of wild-type and mutant NMR studies of albumin are by and are to but with the of and site-directed mutagenesis, of in the are The of the wild-type and mutant is (Fig. but are to two and and in the and for all three mutant that to the two histidines in the mutated zinc His67 and in Fig. also Fig. for a total the for at residues are by Zn2+ binding to most the of and and In the for of to on Zn2+ binding to effects from in the direct effects of Zn2+ or in is that are also on of of zinc Fig. The effects of Zn2+ on the of the mutant Fig. were to those for wild-type consistent with zinc binding to the as by the studies of Asn99 were by protein at K. that is a structural at the interface domains I and II and the of the protein the of albumin in the of mammalian cells, we the of human cells on culture human liver a to cultures. Zn2+ concentrations μm were toxic to cells in with serum Fig. and as by loss of and effects to those of Zn2+ on E. K. J. J. M. 2003; PubMed Scopus Google Scholar, E. S. J.H. A. B. H. J. Clin. 2000; PubMed Scopus Google Scholar). concentrations of μm) were to the effects of Zn2+ on at Zn2+ concentrations as high as μm (Fig. and that the is in and of Zn2+ distribution in cultures. be that the culture used in contains the and mm), which Zn2+ with micromolar affinity K ≈ or ≈ G. and Academic the in Fig. (A and B) are to the of albumin All albumin were also by cells, in the and of The of site-directed mutagenesis, molecular modeling, and has into the structure of the important high-affinity zinc site on human albumin. The residues site are conserved in all mammalian albumin and Fig. except for that of the which domain I human albumin with mutations of of residues are the of site with of affinity but high for likely to to biological transport The coordination of zinc be described as the of His247 is as the sixth ligand. the zinc coordination be as The distances are the for zinc and protein structures D. PubMed Scopus Google Scholar, K. Y. P. J. 2008; Scopus Google Scholar). the is relatively with those for but has in zinc in as an of distances in the K. Y. P. J. 2008; Scopus Google Scholar). that bound be and have a a of zinc Albumin is to but has previously been to and M. Y. K. N. S. T. Otagiri M. 2005; PubMed Scopus Google Scholar). The of zinc albumin has not been possibility be of of with the of as the zinc site in albumin are in in in the the zinc site of a site with and and displays a to that in Fig. site with the of protein is in the in form I. M. M. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). In to albumin, in is no for coordination of a for The form of the is formed only of the which a for In the side of is only as a protein for or for but coordination is during for in the of and such as Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus (86) Google Scholar). In the interdomain zinc site in albumin, Asn99 has a in the of with the of Asn99 of the and likely interdomain in albumin. The that the from albumin of the N99D mutant three for the and with the of NMR for a that interdomain H-bond is important for of the protein The N99D mutation of a by a the interdomain and a and the of His247 Fig. In the N99H mutation an H-bond to be in Fig. is that albumin a for zinc binding in blood but is to that zinc binding in has a on the of albumin, as by the NMR data (Fig. and Fig. The effects of zinc on the NMR of human serum albumin a to the effects by the (see 1986; PubMed Scopus Google and Zn2+ to the of the has been that residues are implicated in the conformational transition J. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). and allosteric effects are important for the of albumin to and transport and M. Curry S. E. M. G. P. S. P. 2005; PubMed Scopus Google Scholar, K. T. K. Y. R. A. Otagiri M. 1999; PubMed Scopus Google Scholar) and have direct on the of W. F. M. Biochem. 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Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1190) Google Scholar). In His247 is to which also an interdomain H-bond to of His67 in albumin. The of Asn99 is an H-bond to the of His247 as the of domains I and II, is not that the binding of a zinc at interface have as is from the NMR We that zinc the interdomain and a of the two be to have an on the of residues at interface, the interdomain in the and of site is of for the and of domains I and a conformational thought to be to the transition also when albumin with J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, T. T. S. S. J. Scholar), and has been that in the of to liver that to the binding of zinc and and is in zinc with in blood have important effects on the transport of of studies show that physiological concentrations of albumin cells from (Fig. at a of of serum albumin the toxic effects of zinc has been shown with S. Y. 2005; PubMed Scopus Google Scholar) and cells E. in 2004; PubMed Scopus Google Scholar). the for a zinc-binding to be in physiological such as blood plasma and the affinity of albumin for zinc is only zinc has been to be of the so-called zinc affinity is likely to the of zinc to The molecular by which is are not in and is that albumin, in to has as a zinc in physiological the of the site is and for zinc and to zinc with the affinity to albumin to and zinc when with

The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.

Albumin has a serum half-life of 3 weeks in humans. This has been utilized to extend the serum persistence of biopharmaceuticals that are fused to albumin. In light of the fact that the neonatal Fc receptor (FcRn) is a key regulator of albumin homeostasis, it is crucial to address how fusion of therapeutics to albumin impacts binding to FcRn. Here, we report on a detailed molecular investigation on how genetic fusion of a short peptide or an single-chain variable fragment (scFv) fragment to human serum albumin (HSA) influences pH-dependent binding to FcRn from mouse, rat, monkey, and human. We have found that fusion to the N- or C-terminal end of HSA only slightly reduces receptor binding, where the most noticeable effect is seen after fusion to the C-terminal end. Furthermore, in contrast to the observed strong binding to human and monkey FcRn, HSA and all HSA fusions bound very poorly to mouse and rat versions of the receptor. Thus, we demonstrate that conventional rodents are limited as preclinical models for analysis of serum half-life of HSA-based biopharmaceuticals. This finding is explained by cross-species differences mainly found within domain III (DIII) of albumin. Our data demonstrate that although fusion, particularly to the C-terminal end, may slightly reduce the affinity for FcRn, HSA is versatile as a carrier of biopharmaceuticals.

Herein we report the use of bromomaleimides for the construction of stable albumin conjugates via conjugation to its native, single accessible, cysteine followed by hydrolysis. Advantages over the classical maleimide approach are highlighted in terms of quantitative hydrolysis and absence of undesirable retro-Michael deconjugation.

Albumin transports both fatty acids and zinc in plasma. Competitive binding studied by isothermal titration calorimetry revealed that physiologically relevant levels of fatty acids modulate the Zn-binding capacity of albumin, with far-reaching implications for biological zinc speciation. The molecular mechanism for this effect is likely due to a large conformational change elicited by fatty acid binding to a high-affinity interdomain site that disrupts at least one Zn site. Albumin may be a molecular device to "translate" certain aspects of the organismal energy state into global zinc signals.

Albumin is an abundant blood protein that acts as a transporter of a plethora of small molecules like fatty acids, hormones, toxins, and drugs. In addition, it has an unusual long serum half-life in humans of nearly 3 weeks, which is attributed to its interaction with the neonatal Fc receptor (FcRn). FcRn protects albumin from intracellular degradation via a pH-dependent cellular recycling mechanism. To understand how FcRn impacts the role of albumin as a distributor, it is of importance to unravel the structural mechanism that determines pH-dependent binding. Here, we show that although the C-terminal domain III (DIII) of human serum albumin (HSA) contains the principal binding site, the N-terminal domain I (DI) is important for optimal FcRn binding. Specifically, structural inspection of human FcRn (hFcRn) in complex with HSA revealed that two exposed loops of DI were in proximity with the receptor. To investigate to what extent these contacts affected hFcRn binding, we targeted selected amino acid residues of the loops by mutagenesis. Screening by in vitro interaction assays revealed that several of the engineered HSA variants showed decreased binding to hFcRn, which was also the case for two missense variants with mutations within these loops. In addition, four of the variants showed improved binding. Our findings demonstrate that both DI and DIII are required for optimal binding to FcRn, which has implications for our understanding of the FcRn-albumin relationship and how albumin acts as a distributor. Such knowledge may inspire development of novel HSA-based diagnostics and therapeutics.

Thymidine phosphorylase (TP) first identified as platelet derived endothelial cell growth factor (PD-ECGF) plays a key role in nucleoside metabolism. Human TP (hTP) is implicated in angiogenesis and is overexpressed in several solid tumors. Here, we report the crystal structures of recombinant hTP and its complex with a substrate 5-iodouracil (5IUR) at 3.0 and 2.5A, respectively. In addition, we provide information on the role of specific residues in the enzymatic activity of hTP through mutagenesis and kinetic studies.

BACKGROUND: Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin. RESULTS: The first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 μm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin. CONCLUSIONS: A Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics.

Albumin is the most abundant protein in blood and plays a pivotal role as a multitransporter of a wide range of molecules such as fatty acids, metabolites, hormones, and toxins. In addition, it binds a variety of drugs. Its role as distributor is supported by its extraordinary serum half-life of 3 weeks. This is related to its size and binding to the cellular receptor FcRn, which rescues albumin from intracellular degradation. Furthermore, the long half-life has fostered a great and increasing interest in utilization of albumin as a carrier of protein therapeutics and chemical drugs. However, to fully understand how FcRn acts as a regulator of albumin homeostasis and to take advantage of the FcRn-albumin interaction in drug design, the interaction interface needs to be dissected. Here, we used a panel of monoclonal antibodies directed towards human FcRn in combination with site-directed mutagenesis and structural modeling to unmask the binding sites for albumin blocking antibodies and albumin on the receptor, which revealed that the interaction is not only strictly pH-dependent, but predominantly hydrophobic in nature. Specifically, we provide mechanistic evidence for a crucial role of a cluster of conserved tryptophan residues that expose a pH-sensitive loop of FcRn, and identify structural differences in proximity to these hot spot residues that explain divergent cross-species binding properties of FcRn. Our findings expand our knowledge of how FcRn is controlling albumin homeostasis at a molecular level, which will guide design and engineering of novel albumin variants with altered transport properties.

A variety of prefractionation methods (including a novel reversed-phase solid-phase-extraction (RP-SPE) combined with SDS-PAGE and proteomic based approaches (e.g., 2-dimensional gel electrophoresis (2DE) and MALDI-TOF mass spectrometry combined with Artificial Neural Network (ANN) bioinformatic tools) were used to investigate the protein/peptide signatures in patients with Polycystic Ovary Syndrome (PCOS). Four potential PCOS biomarkers were identified (complement C4alpha3c and C4gamma and haptoglobin alpha and beta chains).

BACKGROUND: Strict regulation of replisome components is essential to ensure the accurate transmission of the genome to the next generation. The sliding clamp processivity factors play a central role in this regulation, interacting with both DNA polymerases and multiple DNA processing and repair proteins. Clamp binding partners share a common peptide binding motif, the nature of which is essentially conserved from phage through to humans. Given the degree of conservation of these motifs, much research effort has focussed on understanding how the temporal and spatial regulation of multiple clamp binding partners is managed. The bacterial sliding clamps have come under scrutiny as potential targets for rational drug design and comprehensive understanding of the structural basis of their interactions is crucial for success. RESULTS: In this study we describe the crystal structure of a complex of the E. coli β-clamp with a 12-mer peptide from the UmuC protein. UmuC is the catalytic subunit of the translesion DNA polymerase, Pol V (UmuD'₂C). Due to its potentially mutagenic action, Pol V is tightly regulated in the cell to limit access to the replication fork. Atypically for the translesion polymerases, both bacterial and eukaryotic, Pol V is heterotrimeric and its β-clamp binding motif (³⁵⁷QLNLF³⁶¹) is internal to the protein, rather than at the more usual C-terminal position. Our structure shows that the UmuC peptide follows the overall disposition of previously characterised structures with respect to the highly conserved glutamine residue. Despite good agreement with the consensus β-clamp binding motif, distinct variation is shown within the hydrophobic binding pocket. While UmuC Leu-360 interacts as noted in other structures, Phe-361 does not penetrate the pocket at all, sitting above the surface. CONCLUSION: Although the β-clamp binding motif of UmuC conforms to the consensus sequence, variation in its mode of clamp binding is observed compared to related structures, presumably dictated by the proximal aspartate residues that act as linker to the poorly characterised, unique C-terminal domain of UmuC. Additionally, interactions between Asn-359 of UmuC and Arg-152 on the clamp surface may compensate for the reduced interaction of Phe-361.

Post-translational protein modification by the covalent conjugation of ubiquitin, originally implicated as a signal for proteolytic degradation by 26S proteasome, has now been realised to play important roles in the regulation of almost all biological processes in eukaryotes. In order to understand these processes in greater detail there is a requirement for techniques that can purify mixtures of ubiquitin-conjugated proteins, as a prerequisite to their identification and characterisation. Here we review the methods that have been applied to the bulk purification of ubiquitinated proteins and discuss their applications in proteomic analyses of the 'ubiquitome'.

Tissue homeostasis requires a balance between progenitor cell proliferation and loss. Mechanisms that maintain this robust balance are needed to avoid tissue loss or overgrowth. Here we demonstrate that regulation of spindle orientation/asymmetric cell divisions is one mechanism that is used to buffer changes in proliferation and tissue turnover in mammalian skin. Genetic and pharmacologic experiments demonstrate that asymmetric cell divisions were increased in hyperproliferative conditions and decreased under hypoproliferative conditions. Further, active K-Ras also increased the frequency of asymmetric cell divisions. Disruption of spindle orientation in combination with constitutively active K-Ras resulted in massive tissue overgrowth. Together, these data highlight the essential roles of spindle orientation in buffering tissue homeostasis in response to perturbations.

The long circulatory half-life of albumin facilitated by the interaction with the cellular recycling neonatal Fc receptor (FcRn) is utilized for drug half-life extension. FcRn engagement effects following covalent attachment of cargo to cysteine 34, however, have not been investigated. Poly(ethylene glycol) polymers were used to study the influence of cargo molecular weight on human FcRn engagement of recombinant wild type (WT) albumin and an albumin variant engineered for increased FcRn binding. Decreased affinity was observed for all conjugates; however, the engineered albumin maintained an affinity above that of unmodified wild type albumin that promotes it as an attractive drug delivery platform.

BACKGROUND: Baker's yeast Saccharomyces cerevisiae is a proven host for the commercial production of recombinant biopharmaceutical proteins. For the manufacture of heterologous proteins with activities deleterious to the host it can be desirable to minimise production during the growth phase and induce production late in the exponential phase. Protein expression by regulated promoter systems offers the possibility of improving productivity in this way by separating the recombinant protein production phase from the yeast growth phase. Commonly used inducible promoters do not always offer convenient solutions for industrial scale biopharmaceutical production with engineered yeast systems. RESULTS: Here we show improved secretion of the antimicrobial protein, human β-defensin-2, (hBD2), using the S. cerevisiae MET17 promoter by repressing expression during the growth phase. In shake flask culture, a higher final concentration of human β-defensin-2 was obtained using the repressible MET17 promoter system than when using the strong constitutive promoter from proteinase B (PRB1) in a yeast strain developed for high-level commercial production of recombinant proteins. Furthermore, this was achieved in under half the time using the MET17 promoter compared to the PRB1 promoter. Cell density, plasmid copy-number, transcript level and protein concentration in the culture supernatant were used to study the effects of different initial methionine concentrations in the culture media for the production of human β-defensin-2 secreted from S. cerevisiae. CONCLUSIONS: The repressible S. cerevisiae MET17 promoter was more efficient than a strong constitutive promoter for the production of human β-defensin-2 from S. cerevisiae in small-scale culture and offers advantages for the commercial production of this and other heterologous proteins which are deleterious to the host organism. Furthermore, the MET17 promoter activity can be modulated by methionine alone, which has a safety profile applicable to biopharmaceutical manufacturing.

Results An analysis of a series of haploid laboratory yeast strains revealed significant intra-strain variability and unstable plasmid segregation. By combining classic chemical mutagenesis and selection a family of highly efficient Saccharomyces cerevisiae strains has been developed for the commercial production of biopharmaceutical products. When combined with a stable [1], high copy number [2], episomal expression vector system and a strong constitutive promoter, secreted recombinant protein expression titres in excess of 4 g/L were achieved (see Figure 1). Specific genetic modifications to the host were also introduced to increase product yield and control posttranslational modifications, such as proteolysis and glycosylation.

The application of lipases in edible oil processing is a relatively recent development compared to their more general use within food processing. There are two main objectives to enzymatic processing of edible oils. These are to improve an existing process or to allow for the production of unique products that cannot be produced by chemical or other modifications. This chapter discusses application areas for enzyme processing in edible oils. In this application areas, two enzymatic approaches namely enzyme-assisted pressing and total solubilisation of the oil-bearing seed or plant material are used. Enzyme applications post-refining are concerned with modification of the physical properties of the oils, often to change their melting profiles. With the current high cost of both raw materials and energy, the opportunities for yield saving through enzymatic processing in edible oil processing are considerable. Further, enzymatic processes allow a reduction in environmental impact.

This chapter contains sections titled: Introduction Enzyme biochemistry Interesterification Hydrogenation and chemical interesterification Enzymatic interesterification Enzymatic degumming Ester synthesis Speciality fats Environmental benefits of enzymatic processing Future developments in lipase applications Conclusions References