Plasma protein function: Progress and perspectives

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not yet been fully realized, but may have some impact upon pharmacology in xiew of the high degree of drug-binding by this protein. The transport proteins involved in the synthesis and metabolism of haem and haemoglobin (transferrin, haemopexin and haptoglobin) are likely successors to albumin and prealbumin for the fine structure analyses of sequence determination and X-ray crystallography. The proteins which transport lipids in plasma are classified as high. low or very low density, which reflects lipid content. Many of the protein chains associated with these complexes have now been purified and sequenced. A considerable proportion of the polypeptide chain forms the regular ~¢-helix structure and the amino acid sequences indicate that these helices possess well-defined polar and nonpolar surfaces [7]. A model of lipoprotein assembly has been suggested based upon these observations. In this, the n.n-polar underside of the protein helix is embedded in a lipid bilayer o1 micclle-like structure. while the polar surface interacts with the lipid phosphate groups and the aqueous environment [8]. A lipid particle coated with protein agrees with the currently available physical chemical evidence. Many pathological changes involving serum lipoproteins are known; the lesions frequently reflect the absence of protein chains or abnormal protein constituents. In view of recent advances in the structure of lipoproteins, it may be hoped tbat a molecular basis for these pathogies will be determined. The majority of proteins present in plasma contain conjugated sugars. However, for those involved in transport. removal of the carbohydrate portions of the molecules does not have any welldefined effect upon their functions h~ vitro. Aglycosyl transferrin, for example, is able to complex iron as well as does the intact molecule. It must be admitted that despite many hypotheses, the role of carbohydrate attached to any plasma component is not at all clear. One of the most attractive explanations has been made by analogy with the specific blood group sugars. It has been sug~gested that bound carbohydrate represents a coding signal which directs transported molecules to specific receptors in a target tissue. If this is true, it seems strange that no one has yet provided a convincing demonstration. The removal of sialic acid residues from plasma glycoproteins causes their rapid elimination from the circulation. This, at least, is a receptor mediated event; the receptors responsible are on the liver parenchymal cell membranes.

IREVIEWSI Plasma protein function: progress and perspectives M. J. Geisow and A. H. Gordon

ConsMerable advances have been made hi understanding the structure and action O/plasma proteins. Attention is now focused upon the minor components of serum which are recognized as important modulators of defence and repair processes.

The last decade has been a period of rapid development in the field of plasma components. At present more than 200 proteins are known with 70 isolated in a state approaching purity [11. Most important of all, defined functions have been assigned to many of these (Fig. 1) and indications have been obtained for others. In contrast to the earlier classifications of plasma proteins by their electrophoretic mobility, many authors now group components according to function. This emphasizes the large number of components in linked systems, such as complement or blood clotting, which occur in the plasma. Although some plasma proteins have functions in vitro which appear important, individuals who are deficient in these are often relatively unaffected. For example, despite an absence of albumin in their plasma, analbuminaemics remain healthy apart from some mild oedema [2]. In such cases other plasma proteins possibly act in a reserve capacity. This may be true for individuals who lack the so-called thyroxine-binding globulin. Their plasma thyroxine is mainly bound to another thyroidbinding protein called prealbumin [3]. Some of the protease inhibitors of plasma (there are at least six) appear to be similarly redundant when assessed by functions in vitro. So assignments of function based on properties of the isolated proteins may be M.J.G. and A.H.G. are at the National Institute for Medical Research. Mill Hill. London N.W.7, U.K.

misleading; it is.obviously important to consider the role of a component hr vivo, once it has been sufficiently well characterized.

Transport of metabolites Most small molecules, including hormones are bound to proteins in plasma. It is obviously important to bind essential material which would otherwise be lost through filtration in the kidney. However, the specialized environments provided by specific carriers may also be important to protect easily-oxidizable components (vitamin A) or to mobilize poorly-soluble molecules (lipids and steroids). In the last 3 years, the structures and functions of two plasma carriers (albumin and prealbumin) have been worked out in great detail [4.5,6]. The way in which small molecules are bound by these proteins is of some interest, since prealbumin transports the thyroid hormone and also vitamin A as a complex with retinol-binding protein. Albumin, on the other hand, is less specific in its preferences for ligands and will bind a variety of amino acids, drugs and fatty acids. The three-dimensional molecular structure of prealbumin (Fig. 2) possesses two deep, funnel-like depressions into which thyroxine fits. Studies indicate that drugs like aspirin and barbiturates are also bound at these sites, in agreement with clinical observations of high levels of free thyroxine in the plasma of patients treated with aspirin for rheumatism. The implications of the albumin structure have

© Eisevler/North-Holland Biomedical Press 1978

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•FUNCTIONS ENZYMES

PLASMA PROTEINS ENZYME INHIBITORS BINDING PROTEINS

DEFENCE AND REPAIR

Very specific

Less specific

I"Bacteria f Complement system lmmunoglobulins / Viruses ~(minimum of 13 proteins) Interferon A~ . A ~ e . r J Foreign proteins . . . . . . . . ]Blood loss Clotting (minimum of l0 /Raised plasma proteins incl. fibrinogen*) L enzyme l e v e l s ['cxi-Antitrypsin* /Qq-AntichymoJ trypsin* lul-Macroglobulin l Cl-inactivator* I.Antithrombin III Kinin release Kininogenase* Fibrin lysis Plasmin Fibrin stabilization Transglutaminase Muscle relaxant'?. Cholinesterase Antibacterial Lysozyme Transferrin TRANSPORT

Plasma lipoproteins

Cholesterol Esterified fatty acids Phospholipids Cortisol Vitafiain B~t Retinol Haemin Haemoglobin Iron Copper "Aliphatic and aromatic anions Thyroxine

Haptoglobin* (oxidase) Caeruloplasmin* (oxidase)

Transcortin Transcobalamin Retinol-binding protein Haemopexin Haptoglobin* Transfcrrin Caeruloplasmin* Albumin Albumin Thyroxinebinding globulin Albumin Prealbumin*

OTHER

lmmunosuppression Promotion of phagocytosis Antigenicity (HLA antigens)

C-reactive protein* Bt-Microglobulin

~-Fetoprotein ~l-Acid glycoprotein*?

Fig. 1. Functions of the plasma proteins. A classification of plasma proteins according to their functions. Human acute phase proteins are indicated by an asterisk.

Defence and repair processes

It can be seen from Fig. 1 that a considerable proportion of plasma proteins act in a protective capacity. Since many of these are destroyed or modified in the performance of their functions it is hardly surprising that their synthesis is linked to challenges such as tissue injury or infection. In the immune defence system, the mechanism of the greatly increased production of specific antibody in response to an antigenic stimulus is understood in principle. Far less certain are the events which lead to the elevated synthesis of proteins involved in other protective processes, such as fibrinogen and enzyme inhibitors. Several compounds whose level is similarly sensitive to injury do not have any apparent repair function, e.g. prealbumin and caeruloplasmin, Fig. 1. It is apparent that further work in this area is required for a better appreciation of the body's response to various trauma. Apart from the immunoglobuli,~s, all the classical plasma proteins are synthesized

in the liver parenchymal cells. As these cells, in the adult at least, divide extremely slowly and there is no evidence for nervous control, their synthetic output must be under humoral control. This raises questions both of the nature of the stimulatory factors for plasma components and of their cellular origins. Following tissue damage, large numbers of white blood cells gather at the injury site. Tissue debris is removed by these cells and the ensuing phagocytosis stimulates release of many active factors, including newly synthesized proteins. Some of these havi: been reported to lead to the selective stimulation of plasma protein synthesis [9]. Hence for these proteins at least, the origin of the stimulus may be considered to be the plasma leucocytes. It is well known that serum is often an obligatory requirement for cell growth in culture and factors which promote cell lines in defined media have been isolated [10]. Some of these have turned out to be conventional hormones like insulin and

somatomedins (growth factors). Others, distinguished by names like multiplicationstimulating activity remain to be characterized. The molecular weights of some o f these components is in the 15,000-20,000 Dalton range and it is possible that they are themselves intrinsic to plasma, rather than hormonal passengers like insulin. The isolation and investigation of the biochemistry of these humoral factors will undoubtedly be a step of considerable significance in'medicine. An interesting feature of proteins of defensive systems dependent upon the recognition of 'foreign' or 'self" has emerged recently. Immunoglobulins and the membrane-bound tissue antigens have been demonstrated to have close amino acid sequence homology [11], a feature generally accepted to imply structural similarity. Now a further plasma protein has been shown to possess the same homology. This is the C-reactive protein, so called for its ability to precipitate the Cpolysaccharide of pneumococci [12]. Only after injury can this protein be detected in plasma, when it is able to activate the complement system and to promotg phagocytosis by leucocytes. The similar amino acid sequences of these three protein types strongly suggest a common ancestry. It seems likely that they are the descendants of a primitive, undifferentiated host defence system [12]. Plasma enzymes and enzyme inhibitors

Much painstaking work has been invested in unravelling the linked systems of plasma enzymes and their inhibitors. Although incomplete, the major constituents of the three systems responsible for clotting, complement fixation and kinin release have been established. These have been termed 'cascade systems' because of the amplification effect of enzymes activated early in the appropriate system. The complement system must be awarded first prize for complexity because it has

Fig. 2. The polypeptide backbone '~t-carbons only) o f human prealbumin. Thyroxine binds in the large channel in the centre o f the tetrameric )nolecule (T).

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Vol. I, pp. 317-39t, Academic Press, New two alternative pathways and a minimum recently identified I~rostaglandin-synthesis York of'l 8 components, all proteaseS or protease suppressing activity of plasma. The com8 Segrest,J. P., Jackson, R. L., Morrisett, J. D. inhibitors [13]. ponent responsible may be an endogenous and Gotto, A. M. Jr. (1974) FEBS Lett. 38, Clotting starts with the activation of a mediator Of prostaglandin-associated 247 protease known as the Hageman factor effects such as inflammation, through its 9 Mapes, C. A. and ZobocinsM, P. Z. (1977) Am. J. Physiol. 232, CI5-C'22 [14], named after the first individual action on prostaglandin synthesis [18]. recognized as lacking this plasma comConsiderable medical interest already l0 Gospodarowicz, D. and Moran, J. S. (1976) A. Rev. Biochem. 45, 531-558 ponent. After at least four intermediate centres' upon the minor components of II Peterson, P. A., Cunningham, B. A., stages, soluble fibrinogen is converted to plasma. This is particularly true for those Bergghrd, 1. and Edelman, G. M. (1972) fibrin. However, under certain circum- proteins which act as humoral controls of Proc. hath. Aead. Sci. U.S.A. 69, 16q7-1701 stances the clots may be lysed by the plasma protein synthesis. The last 10 years 12 Osmand, A. P., Gewurz, H. and Friedensen, B. (1971) Proc..nam. Acad. Sci. U.S.A. 69, enzyme plasmin formed enzymatically has seen a dramatic increase in knowledge 1697-1701 from plasminogen. Which pathway will of the classical plasma proteins; in the 13 Miiller-Eberhard, J. H. (1975) in The Plasma predominate, clotting or clotting followed ensuing decade the study of these humoral Proteins(Putnam, F. W., ed.), Vol. I, pp. 393by lysis, depends upon the balance of factors will undoubtedly contribute to our 432, Academic Press, New York 14 Austen, D. E. G. and Rizza, C. R. (1974) in enzymic activity and the respective inhi- understanding of disease and recovery. Structure and Ftmction of Plasma Proteins bitors. (Alison, A. W., ed.), Vol. I, pp. 169-193, Of the six protease inbibitors isolated References Plenum Press, London from human plasma, =:antitrypsin is I Schwick, H. G, and Heide, K. (1977) Trends 15 Heimburger, N. 0975) in Proteases and quantitatively the most important and Biochem. Sci. 2, 125-128 Biological Control (Reich, E., R.ifkin, D. B. 2 Gitlin, D. and Gitlin, J. D. 0975) in The and Shaw, E., eds.), Vol. 2, pp.'367-386, Cold represents 90% of the total protease Plasma Proteins (Putnam, F. W., ed.), Vol. 2, Spring Harbor Laboratory, Cold Spring • inhibitory power of plasma [I 5]. Following pp. 330-334, Academic Press, New York Harbor, U.S.A. trauma, proteases are released from injured 3 Tanaka, S. and Starr, P. 0959) J. clin. Endocr. 16 Starkey, P. M. and Barrett, A. J. (1977) in tissue' cells and also from granulocytes and Metab. 19, 485-487 Proteinuses in Mammalian Cells and Tissues macrophages which accumulate at the 4 Brown, J. R. (1975) Fedn Proc. Fedn.Am. Socs (Dingle, J. T., ed.), Vol. 2, pp. 663-696, North exp. Biol..33, 1389 Holland, Amsterdam injury site. Proteases are rapidly com5 Meloun, B., Mor~tvek, L. and Kostka, V. 17 Largman, C., Johnson, J. H., Brodrick, J. W. plexed by ~l-antitrypsin or other inhibi0975) FEBS Lett. 58, 134-137 and Geotzas, M. C. 0977) Nature, Lond. 269, tors, th'us reducing tissue hydrolysis and 6 Blake, C. C. F., Geisow, M. J.i Swann, 168-170 the resulting inflammation. The synthesis I. D. A., Rerat, C. and Rerat, B. (1974) J. 18 Saeed, S. A., McDonald-Gibson, W. J., of =~-antitrypsin and two more.of the six molec. Biol. 88, 1-12 Cuthbert, J., Copas, J. L., Schneider, C., 7 Scanu, A., Edelstein, C. and Keim, P. (19:/5) Gardiner, P. J., Butt, N. M. and Collier, protease inhibitors is increased as a result in The Plasma Proteins (Putnam, F. W., ed.), H. O. J. (1977) Nature, Lond. 270, 32-36 of trauma. Consequently, the balance of proteases and inhibitors is re-establisbed at a higher level following injury. The protease inhibitors vary greatly in their specificity. ~-Antichymotrypsin is strictly specific for chymotrypsin; =smacroglobulin is relatively unspecific and it has been proposed that proteases become entrapped within the inhibitor, as a result of proteolytic attack. A tightlybound protease remains functional, since Joseph Katz and Robert Rognstad small substrates can gain access to the enzyme's active site. Proteins, however, are When interconversion of two compounds occurs by irrerersible reactions, and the enzymes only very slowly cleaved by the trapped catalyzing these reactions are both actire, there is "futile cycling', with dissipation o f energy enzyme. Because hormones the size of without a corresponding change in metaboHtes. There is now evidence for such cycling in insulin can be hydrolysed by a trapped glucose metabolism. How do we measure such cycling? Are futile cycles utile, and i f so, protease, the =~-macroglobulin-complex what is their function? may still play a significant enzymic role in plasma [17]. According to body needs animal cells have there was also extensive lipolys[s, so that It is possible that other enzymes remain the capacity to synthesize and catabolize fatty acids were continuously recycled. unidentified in plasma. It is important a large variety of cell constituents. For Activation of fatty acids to acyI-CoA here to draw a distinction between example, triglycerides are synthesized and esters requires energy, ultimately derived intrinsic and tissue-derived enzymes. The deposited in fat tissue of the fed animal, from ATP, so that recycling dissipates latter are assumed to have arisen by leakage and on fasting are hydrolyzed to glycerol energy without any metabolic gain, and from the tissues. At present only those and fatty acid which are exported via the hence the designation 'futile cycling'. We enzymes or proenzymes associated with the' blood stream. Thus there must be effective have estimated that in epidYdimal fat pad clotting and complement systems are controls of the opposite pathways. Un- tissue of fed rats, the rate of lipolysis is considered to be intrinsic plasma proteins. expectedly, experiments with radioisotopes about one-half that of esterification, and It seems more likely that ,inhibitory in ritro indicated that in adipose tissue that about 10~ of the ATP production is factors remain undiscovered in plasma, simultaneously with active lipogenesis used up in this cycling [1]. There is a host of since these are much less easily recognized J.K. and R.R. are at Cedars-Sinai Medical Center, reactions that potentially could constitute than enzymes. This may be the case for the Los Angeles. CA 90048, U.S.A. futile cycles, but there is experimental ~) Elsevier/North-HollandBiomedicalPress 197S

Futile cycling in glucose metabolism