DIABETES/METABOLISM RESEARCH AND REVIEWS C O M M E N T A R YDiabetes Metab Res Rev 2002; 18: 311–314.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/dmrr.315

Alpha2-HS glycoprotein: a protein in search of afunction

Philippe Arnaud1*Laszlo Kalabay2

1Department of Microbiology andImmunology, Medical University ofSouth Carolina, Charleston, USA2Semmelweis University, Faculty ofMedicine, 3rd Department of InternalMedicine, Budapest, Hungary

*Correspondence to:Philippe Arnaud, Department ofMicrobiology and Immunology,Medical University of SouthCarolina, 171 Ashley Avenue, POBox 250504, Charleston, SC 29405,USA. E-mail: arnaudp@musc.edu

AbstractIn this issue (pp. 305–310) Jun Ren and Amy J. Davidoff author an articleentitled ‘Alpha2-HS glycoprotein, a putative inhibitor of tyrosine kinase,prevents glucose toxicity associated with cardiomyocyte dysfunction’. Theprotein responsible for this biological activity has recently come to theforefront of research on the biological activity of plasma proteins. Copyright 2002 John Wiley & Sons, Ltd.

Keywords alpha2-HS glycoprotein; tyrosine kinase inhibitor; protease inhibitor;biologic function; glucose metabolism

Since its isolation from human plasma in 1960 [1,2], alpha2-HS glycoprotein(AHSG) has been the subject of a number of contradictory reports.

Structure of AHSGOur knowledge about the structure of the human protein has changed overthe years. It is synthesized in the liver [3] and also in osteoblasts [4], andconcentrated into mineralized tissues. If the structure of its sugar moiety iswell established (two N-glycans and three O-glycans [5]), that of the proteinmoiety has been the subject of revisions. It was thought at first to consistof an heterodimer with an A-chain of 282 amino acids and a small B-chainof 27 amino acids, united by a disulfide bridge [6,7]. The isolation of itscDNA followed by its sequence showed that in fact the two chains werepart of a single polypeptide and were united by a 40 amino acid ‘connecting’peptide [8,9]. The processing of the single chain protein into the heterodimer,likely to be due to digestion by chymotrypsin, appears to be mostly a resultof its purification, and would happen in vivo mostly as a consequence ofpathological situations such as sepsis [10]. In addition, the liver protein isphosphorylated, when the bone protein is not [4], and both the single-chainstructure and the phosphorylation appear to be necessary for at least some ofits biological activities.

It is important to note that homologues of AHSG are fetuin [11] inbovines [12] and pp63 in rats [13], and that homologous proteins havebeen sequenced in pig, mouse and sheep [14]. Although the structure ofthe protein is not well conserved (50% homology between human and ratproteins) the position of the 12 cysteine residues (and likely that of thesix resulting disulfide bridges) is highly conserved, stressing the importanceof the secondary structure in the biological activities which are commonbetween species.

Biological roles of AHSGThe biological roles of AHSG, as well as that of its homologues in otherspecies, are equally difficult to clarify. The demonstration of high levels of

Copyright 2002 John Wiley & Sons, Ltd.

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AHSG during the development of several species, as wellas its detection in specific organs (bone marrow, brain,gonads, liver) during their maturation, when it disappearsat or shortly after birth, has lead investigators to proposethat it plays an important role in the development ofthese organs [15–18]. The possibility that fetuin is theactive substance of fetal bovine serum has been thesubject of a number of controversial publications [19].It is likely that the availability of recombinant fetuin (andits homologues) will help to answer this question.

The role of AHSG as a modulator of the immuneresponse has been the subject of numerous reports [20].

Its role in preventing excessive bone mineralization,based upon its concentration in bone tissue, and, morerecently, by the study of a knockout model in mouse, hasnot as yet received a practical application, although twostudies have related the occurrence of variants of AHSGto bone density variations in women [21,22]. Recently,the formation of circulating complexes formed of fetuin,matrix 4-carboxyglutamic acid (GLA) protein and calciumphosphate following inhibition of bone mineralization hasbeen reported [23].

Its role as a protease inhibitor, based upon its structuralanalogy with the cystatins [24], has been discussed foryears. Recent work indicates, nevertheless, that AHSGwould act as an inhibitor of matrix metalloproteinases.

All these properties are thoroughly discussed in anexcellent and recent monograph [25].

In 1989, Cell published an article that postulated anew and fascinating possible role for AHSG. The rathomologue of AHSG (which was not identified as suchthen) called pp63, a phosphoprotein, was found tobe an inhibitor of the activity of the insulin receptor(IR). The characteristics of this effect, abolished bydephosphorylation of the protein, included in vitroinhibition of the tyrosine kinase activity of the receptorand inhibition of its autophosphorylation. In vivo, theprotein was an inhibitor of the growth-promoting effect ofinsulin, although it did not affect other insulin-dependenteffects [13]. Interestingly, IL1b inhibited the synthesis ofthe protein, when insulin did not affect its synthesis, butalmost abolished its phosphorylation [26].

A major step in the extrapolation of these results toman was the expression of recombinant AHSG using thebaculovirus system [27,28].

Recombinant AHSG, 100 times more active than AHSGisolated from human plasma, has been shown to beactive at concentrations of 0.1 mM [29–31], and thisprotein is produced as a fully phosphorylated single-chainpolypeptide [32]. Using this protein, several importantpoints have been demonstrated. AHSG is active on humanas well as on rat insulin receptors, but the activity ofthe preparation decreases with time [31]. The precisemechanism of action of the protein has been partlyelucidated: AHSG binds to the external portion of the IRboth on isolated receptors and on intact cells. It does notcompete with the binding of insulin. The phosphorylationof IRS-1 is also abolished The effect of AHSG on the IR

and IRS-1 are also observed in vivo in the liver and musclefollowing injection of the protein in intact rats [30,33].

Finally, two other examples are of interest in discussinga possible role of IR modulation by AHSG. First, a geneticmodel of rat obesity shows an increased expression of theAHSG gene as evidenced by differential display followinga high fat diet; in contrast, normal rats do not raisetheir AHSG expression [34]. Second, in humans, one ofus (Kalabay et al., in press) has shown that patients withgestational diabetes have higher than normal levels ofAHSG. These levels correlate positively with leptin levelsand indirect parameters of insulin resistance.

In this view it is tempting to speculate that AHSGdoes indeed represent a physiological modulator of theIR activity. It is important to stress, in this respect, thatpathological changes of AHSG concentration occur as itis one of the rare negative acute phase proteins whoselevels are substantially reduced following infection andinflammation [35]. This decrease is controlled by severalcytokines, which act directly on the expression of thegene through its promoter region [36,37]. Finally, AHSGis a polymorphic protein [38,39], and two of the severalalleles reported are present in all populations studied atpolymorphic frequencies [40].

Nevertheless, it should be borne in mind that the aminoacid substitutions between these alleles are minute [41]when compared to the amino acid substitutions foundbetween two species [14], where the biological effectappears to be of the same order of magnitude (the effectsof bovine versus human recombinant fetuin on insulinreceptors are identical [30,33,42]).

Nevertheless, at least two important questions need tobe answered. First, how can we explain that AHSG-fetuin,present in normal plasma at concentrations 100 timeshigher that those effective in blocking IRs in vitro, does notappear to present such an effect in normal circumstances?A possibility is that the liver form, produced as a fullyactive protein, becomes rapidly dephosphorylated and/orprocessed, and that the form found in normal plasmais over 90% inactive. Second, it is difficult to explain,with the present state of our knowledge, how this proteinaffects only the mitogenic pathway of the IR withoutaltering the other functions (especially the metabolicfunctions) associated with its activation, bearing in mindthat AHSG is shown to block the primary steps in insulinsignaling, mainly IR autophosphorylation and tyrosinekinase activity, as well as IRS-1 phosphorylation? It ispossible that AHSG could also antagonize the effect ofother growth factors and cytokines. In this respect it isworth noting that the first cystatin domain of AHSG bindsto TGF-b and TGF-related bone morphogenic protein, asa result blocking their antiproliferative action [43]. Hereagain a deeper knowledge of the exact mechanisms ofIR signal transduction [44] should allow this importantquestion to be solved.

The work of Ren and Davidoff, as well as other workbeing conducted at the present time, such as a thoroughanalysis of glucose metabolism in knockout mice [33], will

Copyright 2002 John Wiley & Sons, Ltd. Diabetes Metab Res Rev 2002; 18: 311–314.

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certainly help to shed light on the biological functions ofthis fascinating protein.

Acknowledgements

The present work was supported in part by grants from LabcatalLaboratories, Montrouge, France and from The AlternativesResearch & Development Foundation, Eden Prairie, MN 55346-3000, USA.

References

1. Heremans JF (ed.). Les Globulines Seriques du Systeme Gamma.Arscia: Brussels, 1960; 103–105.

2. Schmid K, Burgi W. Preparation and properties of the humanplasma Ba-alpha2 glycoprotein. Biochim Biophys Acta 1960; 17:440–453.

3. Putnam FW. Progress in plasma proteins. In The Plasma Proteins,vol. 4. Putnam FW (ed.). Academic Press: New York, NY, 1984;1–44.

4. Ohnishi T, Arakaki N, Nakamura O, Hirono S, Daikuhara Y.Purification, characterization, and studies on biosynthesis ofa 59-kDa bone sialic acid-containing protein (BSP) from ratmandible using a monoclonal antibody. Evidence that 59-kDa BSP may be the rat counterpart of human alpha2-HSglycoprotein and is synthesized by both hepatocytes andosteoblasts. J Biol Chem 1991; 266: 14 636–14 645.

5. Hayase T, Rice KG, Dziegielewska KM, Kuhlenschmidt M,Lee YC. Comparison of N-glycosides of fetuins from differentspecies and human alpha2-HS glycoprotein. Biochemistry 1992;31: 4915–4921.

6. Gejyo F, Chang JL, Burgi W, et al. Characterization of the B-chain of human plasma alpha2-HS glycoprotein. J Biol Chem1983; 258: 4966–4971.

7. Gejyo F, Schmid K. Purification and characterization of thetwo forms of human plasma alpha2-HS glycoprotein. BiochimBiophys Acta 1981; 671: 78–84.

8. Arnaud P, Mietz JA, Grossman Z, McBride OW. Isolation andcharacterization of a cDNA clone for human alpha2-HSglycoprotein. In Proteins in Biological Fluids, vol. 35. Peeters H(ed.). Pergamon Press: Oxford, 1987; 135–138.

9. Lee CC, Bowman BH, Yang F. Human alpha2-HS glycoprotein:the A and B chains with a connecting sequence are encoded bya single mRNA transcript. Proc Natl Acad Sci U S A 1987; 84:4403–4407.

10. Jahnen-Dechent W, Trindl A, Godovac-Zimmermann J, Muller-Esterl W. Posttranslational processing of human alpha2-HSglycoprotein (human fetuin). Evidence for the production ofa phosphorylated single-chain form by hepatoma cells. Eur JBiochem 1994; 226: 59–69.

11. Pedersen KO. Fetuin, a new globulin isolated from serum. Nature1944; 154: 575.

12. Dziegielewska KM, Brown WM, Casey SJ, et al. The completecDNA and amino acid sequence of bovine fetuin. Its homologywith alpha2-HS glycoprotein and relation to other members ofthe cystatin family. J Biol Chem 1990; 265: 4354–4357.

13. Auberger P, Falquerho L, Contreres JO, et al. Characterization ofa natural inhibitor of the insulin receptor tyrosine kinase: cDNAcloning, purification, and anti-mitogenic activity. Cell 1989; 58:631–640.

14. Brown WM, Dziegielewska KM, Saunders NR, Christie DL,Nawratil P, Muller-Esterl W. The nucleotide and deduced aminoacid structures of sheep and pig fetuin. Common structuralfeatures of the mammalian fetuin family. Eur J Biochem 1992;205: 321–331.

15. Terkelsen OBF, Jahnen-Dechent W, Nielsen H, et al. Rat fetuin:distribution of protein and mRNA in embryonic and neonataltissues. Acta Embryol 1998; 197: 125–133.

16. Dziegielewska KM, Matthews N, Saunders NR, Wilkinson G.Alpha2-HS glycoprotein is expressed at high concentration inhuman fetal plasma and CSF. Fetal Diagn Ther 1992; 8: 22–27.

17. Dziegielewska KM, Brown WM, Deal A, Foster KA, Fry EJ,Saunders NR. The expression of fetuin in the development and

maturation of the hemopoietic and immune systems. HistochemCell Biol 1996; 106: 319–330.

18. Dziegielewska KM, Daikuhara Y, Ohnishi T, et al. Fetuin in thedeveloping neocortex of the rat: distribution and origin. J CompNeurol 2000; 423: 373–388.

19. Nie Z. Fetuin: its enigmatic property on growth promotion. AmJ Physiol 1992; 263: C551–C562.

20. Lewis JG. Alpha2-HS glycoprotein: functional studies. PhDThesis, University of Otago, Dunedin, New Zealand, 1983.

21. Eichner JE, Friedrich CA, Cauley JA, et al. Alpha2-HSglycoprotein phenotypes and quantitative hormone and bonemeasures in postmenopausal women. Calcif Tissue Int 1990; 47:345–349.

22. Dickson IR, Gwilliam M, Arora M, et al. Lumbar vertebral andfemoral neck bone mineral density are higher in postmenopausalwomen with the alpha2-HS glycoprotein 2 phenotype. BoneMiner 1994; 24: 181–188.

23. Price PA, Thomas GR, Pardini AW, Figueira WF, Caputo JM,Williamson MK. Discovery of a high molecular weight complexof calcium, phosphate, fetuin and matrix g-carboxyglutamic acidprotein in the serum of etidronate-treated rats. J Biol Chem 2002;277: 3926–3934.

24. Kellermann J, Haupt H, Auerswald EA, Muller-Esterl W. Thearrangement of disulfide loops in human alpha-2-HSglycoprotein – similarity to the disulfide bridge structuresof cystatins and kininogens. J Biol Chem 1989; 264:14 121–14 128.

25. Dziegielewska KM, Brown WM. Fetuin. Springer-Verlag:Heidelberg, 1995.

26. Akhoundi C, Amiot M, Auberger P, LeCam A, Rossi B. Insulinand interleukin-1 differentially regulate pp63, an acute phasephosphoprotein in hepatoma cell line. J Biol Chem 1994; 269:15 925–15 930.

27. Kalabay L, Arnaud P. The production of human recombinantalpha2-HS glycoprotein in the baculovirus vector system. In22nd Congress of the International Society of Internal Medicine,Varro V, DeChatel R (eds). Monduzzi Editore SPA: Bologna,1994; 613–616.

28. Srinivas PR, Goustin AS, Grunberger G. Baculoviral expressionof a natural inhibitor of the human insulin receptor tyrosinekinase. Biochem Biophys Res Commun 1995; 208: 879–885.

29. Srinivas PR, Wagner AS, Reddy LV, et al. Serum alpha2-HSglycoprotein is an inhibitor of the human insulin receptor atthe tyrosine kinase level. Mol Endocrinol 1993; 7: 1445–1455.

30. Srinivas PR, Deutsch DD, Mathews ST, Goustin AS, Leon MA,Grunberger G. Recombinant human alpha2-HS glycoproteininhibits insulin-stimulated mitogenic pathway without affectingmetabolic signalling in Chinese hamster ovary cellsoverexpressing the human insulin receptor. Cell Signal 1996;8: 567–573.

31. Kalabay L, Chavin K, Lebreton JP, Robinson KA, Buse MG,Arnaud P. Human recombinant alpha2-HS glycoprotein isproduced in insect cells as a full length inhibitor of theinsulin receptor tyrosine kinase. Horm Metab Res 1998; 30:1–6.

32. Kalabay L, Mathur S, Bobin S, Arnaud P. Electrophoretic andisoelectric focusing analysis of human recombinant alpha2-HSglycoprotein produced in insect cells: analysis of the post-translational events. Electrophoresis 1996; 17: 529–532.

33. Mathews ST, Chellam N, Srinivas PR, et al. Alpha2-HSg, aspecific inhibitor of insulin receptor autophosphorylation,interacts with the insulin receptor. Mol Cell Endocrinol 2000;164: 87–98.

34. Lin X, Braymer HD, Bray GA, York DA. Differential expressionof insulin receptor tyrosine kinase inhibitor (fetuin) gene in amodel of diet-induced obesity. Life Sci 1998; 63: 145–153.

35. Lebreton JP, Joisel F, Raoult JP, Lannuzel B, Rogez JP,Humbert G. Serum concentration of human alpha2-HSglycoprotein during the inflammatory process. Evidence thatalpha2-HS glycoprotein is a negative acute-phase reactant. JClin Invest 1979; 64: 1118–1129.

36. Daveau M, Davrinche C, Julen N, Hiron M, Arnaud P,Lebreton JP. The synthesis of human Alpha2-HS glycoproteinis down-regulated by cytokines in hepatoma Hep G2 cells. FEBSLett 1988; 241: 191–194.

37. Daveau M, Davrinche C, Djelassi N, et al. Partial hepatectomyand mediators of inflammation decrease the expression of liveralpha-2-HS glycoprotein gene in rats. FEBS Lett 1990; 273:79–81.

Copyright 2002 John Wiley & Sons, Ltd. Diabetes Metab Res Rev 2002; 18: 311–314.

314 Commentary

38. Anderson NL, Anderson NG. Microheterogeneity of serumtransferrin, haptoglobin and alpha2-HS glycoproteinexamined by high resolution two-dimensional elec-trophoresis. Biochem Biophys Res Commun 1979; 88:258–265.

39. Boutin B, Feng SH, Arnaud P. The genetic polymorphism ofalpha2-HS glycoprotein: study by ultrathin layer isoelectricfocusing and immunoblot. Am J Hum Genet 1985; 37:1098–1105.

40. Arnaud P, Miribel L, Roux AF. Alpha2-HS glycoprotein. InMethods in Enzymology, Immunochemical Techniques, Part H:Acute-phase Reactants, vol. 163. Sabato GD (ed.). AcademicPress: London, 1988; 431–441.

41. Osawa M, Umetsu K, Ohki T, Nagasawa T, Suzuki T,Takeichi S. Molecular evidence for human alpha2-HSglycoprotein (AHSG) polymorphism. Hum Genet 1997; 99:18–21.

42. Mathews ST, Srinivas PR, Leon MA, Grunberger G. Bovinefetuin is an inhibitor of insulin receptor tyrosine kinase. LifeSci 1997; 61: 1583–1592.

43. Demetriou M, Binkert C, Sukhu B, Tenenbaum HC, Dennis JW.Fetuin/alpha2-HS glycoprotein is a transforming growth-b typeII receptor mimic and cytokine antagonist. J Biol Chem 1996;271: 12 755–12 761.

44. White MF, Kahn CR. The insulin signaling system. J Biol Chem1994; 269: 1–4.

Copyright 2002 John Wiley & Sons, Ltd. Diabetes Metab Res Rev 2002; 18: 311–314.