a2-Macroglobulin, a multifunctional bi#{252}ding

protein with
targeting characteristics
WOLFGANG BORTH’
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA

ABSTRACT a2-macroglobulin (a2M) and related proteins cyclic thiol ester bond, they have also been termed the thiol
share the function of binding host or foreign peptides and ester plasma protein family. C5 is a remote member of the
particles, thereby serving as humoral defense barriers aMs and lacks the thiol ester. In humans, aMs comprise as
against pathogens in the plasma and tissues of ver- much as 8-10% of total serum protein. Proteins homologous
tebrates. In human ct2M, several reactive sites including to human a2M are present in the circulation of vertebrates
high-affinity sites for zinc, transglutaminase cross-linking (sometimes in multiple forms, as in some rodents) and inver-
sites, and reactive sites derived from the activated thiol tebrates as well as in the egg white of birds and reptiles.
ester can mediate reversible or irreversible capture of pro- Homologues of a2M can be traced to arthropods, e.g., the
teins of diverse biological functions. cr2M interacts and American horseshoe crab (Limulus polyp/iemus), and thus
captures virtually any proteinase whether self or foreign, emerged more than 550 million years ago when the arthro-
suggesting a function as a unique “panproteinase inhibi- pod and vertebrate lineages diverged (3, 4). In body fluids,
tor.” Activation of a2M generates novel binding sites, a2M homologues circulate as monomers (i.e., a,-inhibitor 3
which mediate complex formation with cytokines and in rat and hamster, murinoglobulin in mice), dimers (i.e.,
other peptides. Direct evidence of physical association of PZP and invertebrate a2Ms), and tetramers (most a2Ms in
cytokines with activated a2M indicated its role as bio- circulation and those found in eggs) composed of identical
logical response modifier in cell cultures. A mechanism subunits.
commonly referred to as “clearance of activated ct2M” in-
volves Ca2-dependent binding to a specific cell surface THE BINDING FUNCTION OF a2M
receptor, a member of the low-density lipoprotein recep-
tor supergene family, that mediates cellular uptake by en- Multiple reactive sites concealed within a2M suggest
docytosis and delivery to endosomes and lysosomes. The diversified and complex functions as a binding, carrier, and
peptide binding function of a2M, therefore, may also be targeting protein. In human a2M, a 720-kDa glycoprotein,
viewed as a mechanism that allows targeting of biologi- five reactive sites have been characterized in each of its four
cally active peptides to different cell types expressing the identical subunits (180 kDa) (Fig. 1). 1) The bait region: this
a2M receptor. Internalized complexes may be dispatched is an exposed 25 amino acid stretch within the middle of each
into different pathways of endocytic/lysosomal pathways subunit that can be cleaved by virtually any proteinase,
in a cell type-specific manner. In addition, bioactive pep- whether originating in the host or released either by microor-
tides bound to ct2M may dissociate in the process of in- ganisms or as a venom (5-8). Bait region cleavage results in
tracellular ligand sorting, thereby modulating cell func- an immediate and distinct conformational change of the
tion, or remain bound and share the catabolic fate of a2M molecule that is intimately linked with the cleavage of
a2M. The diversified and probably programmed binding the internal thiol ester. The instantaneous conformational
functions of a2M indicate that in addition to its role in change mediates ‘trapping” of the proteinase and physical
trapping proteinases, it has other biological activities that exclusion of large (but not small) substrates from the active
remain to be fully defined. That a2M may function as a center of the enzyme. Thus, the trapping of a proteinase by
binding and carrier protein with targeting characteristics a2M, generally referred to as “inhibition,” does not block the
is predicted from 1) the known functions of cs2M, and 2) proteinase’s active site, and thus contrasts with any other
the similarity of the fate of a2M with proteins whose known mechanism of proteinase inhibitor-proteinase com-
significance in targeting and intracellular trafficking has plex formation that involves irreversible active site inhibi-
been studied in more detail. - Borth, W. a2-Macroglo- tion. Vice versa, nonproteolytic peptides trapped by a2M be-
bulin, a multifunctional binding protein with targeting come largely protected from exogenous proteinases.
characteristics. FASEB J. 6: 3345-3353; 1992. Monomeric aMs fail to trap proteinases and no covalent

Key Wordc: cytokines lipoprotein receptor disulfid.e bonds thiol

ester toxins . endocytosis processing
‘Address for correspondence: Division of Hematology, Mount
Sinai School of Medicine, Box 1079, One Gustave L. Levy Place,
a-MACROGLOBULINS OR THIOL ESTER PLASMA New York, NY 10029-6574, USA.
PROTEINS 2Abbreviations: a2M, a2-macroglobulin; PZP, pregnancy zone
protein; aMs, a-macroglobulins; Ab, monoclonal antibody;
a2MR, a2M receptor; TGs, transglutaminases; IL-6, interleukin-6;
cxm-Macroglobulin (cs2M)2 is the major representative of a al-Inh 3, al-inhibitor III; LRP, lipoprotein receptor-related pro-
group of plasma proteins that include complement compo- tein; IT, immunotoxin; PDGF, platelet-derived growth factor; TGF-
nents C3 and C4 as well as pregnancy zone protein (PZP). fi, transforming growth factor-fl; bFGF, basic fibroblast growth fac-
This protein family is collectively called a-macroglobulins tor; NGF, nerve growth factor; D-PA, D-penicillamine; RAP,
(aMs) (1, 2). Because these proteins have a unique internal receptor-associated protein.

0892-6638/92JcXO6-3345/$01.50. © FASEB 3345

 , . Monoclonal antibodies (Ab’s) raised against this site block
,aII
‘4 #{149};i
coou
receptor binding (15). The a2M receptor (a2MR) is a
600-kDa cell surface glycoprotein (16, 17) and a unique
member of the low density lipoprotein receptor superfamily
(18, 19). 4) The transglutaminase reactive site: transglutaminases
(TGs), whether of tissue origin (tissue TGs) or in plasma
SaM
‘ I (plasma TG = coagulation factor XIII), catalyze incorpora-
reduct,t tion of primary amines into protein substrates as well as for-
p,ote,nases
0 mation
between
of irreversible
susceptible
e(’y-glutamyl)lysine
protein substrates
isopeptides
(20). Tissue
bonds
trans-
glutaminases are abundantly produced by numerous cell
Fa2M
types including macrophages, keratinocytes, and endothelial
cells. Active ‘ID is found on cell surfaces and in tissues where
4
p
0 it associates
substrate
with matrix components.
in human serum (21). A ‘ID-reactive
a2M is the major TG
site in cx2M
.1 is accessible in the native molecule and is in close proximity
,,
to the bait region (approximately 20 amino acids upstream
Figure 1. The human a2M and its reactive sites. Accessibility of from the primary proteinase cleavage site in the bait region)
reactive sites and conversion of native (Sa2M) to the activated (1). TGs catalyze the incorporation of primary amines and
(Fa,M) a2M (modified after refs 1, 2, 5, 7, 8). Proposed position peptides (22) into a2M. 5) a2M is a metalloprotein and is a
in hierarchy of the binding or cross-linking sites in a monomeric major zinc binding plasma protein (23). Zinc is not required
a2M subunit (180 kDa) is indicated by circled number (1-4): the for the proteinase binding function of a2M, but is required
transglutaminase reactive site (amino acids (aa) 670 and 671) is the for the binding of IL-1f3 (12). A detailed review of the ultras-
first obligatory reactive site; step four, the receptor binding site tructure on a2M and a tentative three-dimensional model
(RBS, aa 1313-1451), is the last site, accessible only after cleavage of are discussed elsewhere (24).
the thiol ester (aa 949-952) in step 3. Step 2, the cleavage of the bait
region (aa 675-700) by proteinases that results in cleavage of the
thiol ester, can be by-passed by the use of amines or reductants that SYNTHESIS OF a2M
directly cleave the thiol ester by nucleophilic attack. Cleavage also
occurs upon heating or prolonged storage of native a2M. The trans- The a2M gene is a single-copy gene in the human genome
glutaminase reactive site and the bait region are accessible in and belongs to a gene cluster comprising the authentic a2M
Sa2M, while the cytokine binding site and receptor binding site be- gene, an a2M pseudogene, and the PZP gene on human
come accessible in Fa2M. Proposed ligands are, Rl = TG-reactive chromosome l2pl2-13 (25). Mutations within the bait region
peptide, R2 = proteinase, R3 = cytokine, toxin. Zinc binding sites or other regions have been described, but no functional alter-
are not shown. ations in plasma a2M were seen (26). The potential physio-
logical consequences of a2M deficiency are not known. The
absence of total deficiency in humans supports the notion of
cupture of proteinases is accomplished if proteinases lack ac- the fundamental function of a2M. As a2M is synthesized by
cessible lysines (e.g., neutrophil elastase). Therefore, mono- several cell types, it is conceivable that in tissues a2M gene
meric aMs can be activated by proteinases, which does not expression is under the control of cell type-specific regulatory
result in complex formation with proteinases. 2) The internal elements. Several transcription initiation sites have been
thiol ester: the possibility that an internal thiol ester could identified. The 5 flanking region of exon 1 of the a2M gene
form in proteins was proposed in 1942 (9). Forty years later contains regulatory sequences homologous to the IL-6
a family of proteins was identified with the proposed struc- response element as well as to the HP-I element, a metal
ture. The linkage formed between a cysteine and glutamine responsive element, and a potential Spi binding site.
is labile and readily cleaved by heat (10), small nucleophiles Numerous cell lineages, including lung fibroblasts,
including primary amines, reductants, and water. Activation monocytes-macrophages, hepatocytes, and astrocytes, syn-
of a2M by methylamine is frequently used in experimental thesize a2M (1, 2, 25). Some tumor cell lines are capable of
settings, but in vivo this is accomplished by proteinases. Ac- producing significant amounts of a2M. Perhaps the best-
tivation of a2M by proteinases produces an intermediate studied example of quantitative differences in a2M synthesis
(‘nascent”) a2M (11) with reactive glutamyl and cysteinyl in vivo and in vitro are melanoma cells (27). Immuno-
residues in each of the four subunits. The reactivity of the histochemistry of primary tumors and their metastases
glutamyl residue decays rapidly by reacting with water, but showed that approximately 33% of melanoma cells have in-
is also capable of covalent reaction with lysine residues of an tracellular staining for a2M which suggests a2M synthesis by
adjacent peptide, including those of the attacking proteinase these cells. Clones established from various melanoma cell
or nonproteolytic peptides present during the reaction. The lines produced and secreted a2M that accounted for as much
cysteinyl residue remains reactive and can bind cytokines as 50% of total protein production by these cells. Synthesis
(12) and the A chain of the plant toxin ricin (13). Because of a2M by several normal tissues is under humoral control.
cleavage of the internal thiol ester by proteinases or In cultured bovine adrenocortical cells, synthesis of a2M
nucleophilic compounds leads to an electrophoretic shift may be selectively and significantly increased by TGF-/3 (28).
from a slow to faster form in native polyacryl-amide gel dcc- During cell differentiation in the rat ovary, a2M is regulated
trophoresis (7), the literature has acquired the term Sa2M by luteinizing hormone and prolactin (29). In cultured liver
for the native a2M and Fa2M (or a2M) for its activated fat sorting (Ito) cells, dexamethasone increases steady-state
form. 3) The receptor binding site: this site comprising the levels of azM-specific messenger RNA and decreases
COOH-terminal 138 amino acids is expressed in each of the apolipoprotein E-specific transcripts, but not those of
four subunits after cleavage of the thiol ester (14). fibronectin and actin. (30). Interleukin-6 (IL-6) induces

3346 Vol. 6 December 1992 The FASEB Journal BORTH

a2M synthesis by human neuronal cells, a mechanism of tein = RAP) and appears to regulate the receptors affinity
possible significance in some diseases of the central nervous for itsligands (42). a2MR/LRP is a multidomain receptor
system (31). In humans, however, a2M is not considered an containing terminal complement-type repeats, which likely
acute-phase protein (6), but in rodents aM homologues are are the ligand binding domains, and epidermal growth fac-
known to be regulated by acute-phase mediators (32). A link tor precursor homology domain repeats that undergo an
between cholesterol metabolism and regulation of a2M gene acid-inducible conformational change, allowing the ligand to
transcription was shown in an animal model (33). In ham- dissociate from the receptor upon acidification of endo-
sters, a cholesterol-rich diet suppresses a 1-inhibitor III somes. Other ligands for a2MR/LRP thus far characterized
(a,-Inh 3) mRNA as well as plasma a1-Inh 3 protein levels. include apolipoprotein E-rich chylomicron remnants (43).
The al-Inh 3 gene encodes a member of the a2M family in More recently this receptor was also shown to bind and in-
the rat and hamster. Drugs that lower plasma cholesterol ternalizespecifically urokinase . plasminogen activator inhi-
levels increased a 1-Inh 3 mRNA levels.This observation bitor type-i complexes (44) as well as Pseudomonas aeruginosa
suggested that the regulation of the al-Inh 3 gene by exotoxin A (45). The affinityof cr2MR/LRP for ligands of
cholesterol parallels that of other genes that maintain diverse biologicalfunctions could imply that this receptor
cholesterol homeostasis, including 3-hydroxy-3-methylglutaryl has various biological roles.a2MR has been identifiedon
coenzyme A reductase and synthase as well as low density fibroblasts, hepatocytes, adipocytes, syncytiotrophoblasts,
lipoprotein receptor. astrocytes, monocytes, and macrophages (46). a2MR/LRP
Synthesis and cell surface expression of the a2M receptor isdefined as a monocyte differentiationantigen because itis
is also under humoral control and is regulated by insulin in absent on other blood cells. Flow cytometry analysis showed
adipocytes (34) and CSF-1 in mouse bone marrow macro- that a2MR appears at an early stage of monocyte differentia-
phages (35). Therefore, itis conceivable that tissue-specific tion and is frequently seen as a marker in chronic my-
a2M synthesis as well as uptake reflects a compartmentalized elomonocytic leukemias (47). Fibroblasts and other cells of
function of a2M that is independent of plasma a2M. A possi- mesenchymal origin have been widely used for in vitro bind-
ble role of cell secretion of a2M might be to regulate tissue ing studies, but the role of these cells in Fa2M clearance in
proteinases such as matrix metalloproteinases as well as vivo lacks evidence. Immunohistochemical studies of tissues
cytokines in the extracellularmatrix, where access from the as well as clearance studiesusing labeled Fa2M consistently
plasma would be limited (36, 37). showed that uptake of labeled Fa2M is exercised by tissue-
A significant increase in a2M plasma levels is consistently fixed macrophages in the liverand synovial tissuesas well as
seen during embryogenesis, pregnancy, and childhood, all other tissuesand hepatocytes (41, 48, 49).
representing periods of life characterized by growth, de- Homologues of a2MR/LRP and a2M evolved in the
velopment, and differentiation (1, 2, 6, 38, 39). PZP, the chicken, but thisspecies does not produce apolipoprotein E.
closest homologue of a2M in the human, is present in trace In the laying hen, at least three a2M binding receptors, all
amounts in normal plasma, but is significantly elevated in related to the low density lipoprotein receptor superfamily,
the sera of pregnant women where its function is unknown. have been identified: one that is somatic and two that are
Activated PZP shares the same receptor as activated a2M. oocyte-specific (50, 51). In the oocyte these receptors mediate
The significance of PZP as a proteinase inhibitor is enig- import of the major yolk precursors, very low density
matic, as it binds only a narrow range of proteinases (1), in- lipoprotein,and vitellogenin.Because in ligand blot studies
dicating that it has lost the panproteinase inhibitor qualities of a2M binds specifically to all three receptor forms, it is pro-
a2M; itscytokine binding quality remains unimpaired (40). posed that these receptors may also have a function in a2M
transport and perhaps help load chicken egg white a2M,
called ovostatin, into the egg.
RECEPTOR-MEDIATED ENDOCYTOSIS OF The association of activated a2M with its receptor is
ACTIVATED a2M highly pH-dependent, and acidification to pH 6.8 abolishes
ligand receptor interaction(52).This suggests that upon en-
It has been recognized since 1971 that a2M-proteinase com- docytosis a2M dissociates at relatively mildly acidic condi-
plexes are rapidly cleared from the circulation, indicating the tions early in the pathway of intracellular ligand sorting.
presence of a specific receptor for the proteinase-activated This contrasts with other ligands undergoing receptor-
form of a2M (41).Almost 20 years later itbecame evident mediated endocytosis, e.g., transferrin or proteins bearing
that the a2MR is identical with the low-density lipoprotein the mannose-6-phosphate recognition marker, that are
receptor-related protein (LRP) (16-19). The a2MR/LRP released from their receptors at a pH of 6 or less. The lower-
gene may represent the ancestral gene from which the LDL ing of pH in endosomes also creates an optimal milieu for the
receptor gene evolved by duplication and deletions of the activity of endosomal proteinases, which may vary in endo-
LRP gene. A gene equivalent to a2MR/LRP was cloned somes of different cell types, indicating differences in
from the primitive roundworm Caenorhabditis elegaru, but the processing adapted to the cell’s function (53). In addition,
LDL gene was observed only in higher developed animals endosomes (54, 55) and lysosomes (56) are capable of reduc-
early in the evolution of vertebrates. Thus, early evolution of tivecleavage of disulfidebonds in peptides. This endosomal
a2M and its receptor appears to be closely linked in inver- processing step is thought to be crucial for toxin activation
tebrates, whereas a2MR/LRP-related receptors (i.e., LDL (57, 58) and to precede macromolecular degradation (54, 55)
receptor) appeared later with the vertebrates. Some ligands and antigen processing (56). Recent evidence indicates that
for a2MR/LRP (including apolipoprotein E) have evolved reductive activation of some toxins is catalyzed by sulf-
only in mammals. The a2MR/LRP is a 600-kDa hydryls present on cell surfaces. Upon internalization of
glycoprotein that undergoes proteolysis in the trans-Golgi and ligands, membrane-associated sulfhydryls also act within en-
is expressed as a noncovalently associated heterodimer of 515 dosomes at various stages of ligand routing and/or sorting.
and 85 kDa, respectively. A third protein, a 39-kDa It has been proposed that likely candidates for membrane-
glycoprotein with a heparin binding domain, can be as- associated reduction are members of the thioredoxin-
sociated with the receptor (receptor-associated pro- thioreductase family, a highly conserved and virtually ubiq-

BIOLOGY OF a2M 3347

uitous oxidoreductase enzyme system (59, 60). Internalization tivating a2M in the presence of a-galactosidase and feeding
and degradation of a2M vary considerably with the cell type the cells with the activated a2M that had entrapped the en-
studied. It can be rapid with a t112 of 15 min or slow zyme. A significant increase in intralysosomal galactosidase
with a t112 of I h. Intact a2M may also be sequestered for 24 activity was recorded that resulted from receptor-mediated
h and significant amounts of a2M may be regurgitated intact endocytosis of these a2M complexes (66).
(52, 61, 62). Internalization of a2M is abrogated in fibrob- The mechanism of receptor-mediated endocytosis and in-
lasts after transformation with Maloney sarcoma virus (63). tracellular activation is also used by plant toxins that exert
Monoclonal Ab’s directed against either the 515- or 85-kDa their cytotoxic effects after receptor-mediated uptake of the
subunit of a2MR/LRP are rapidly endocytosed by either latent toxin, reductive and/or proteolytic activation within
subunit, but only the Ab against the larger subunit becomes an endocytic compartment, and translocation to the cytosol,
degraded whereas the Ab against the smaller subunit recy- thereby gaining access to and interfering with vital cellular
cles intact to the cell surface (64). This indicates that the two functions (13). For example, the uptake of the toxin ricin is
subunits of a2M/LRP have different, perhaps programmed, mediated by one of its two disulfide-bonded subunits - the B
competences and/or functions in the sorting and dispatching chain - but it is the A chain that mediates the cytotoxic effect
process in endosomes. It isconceivable that the intracellular after reductive release from the B chain within the cell.
fateof a2M complexes isdictatedby the celllineitenters and Another toxin, Pasteurella multocida toxin, functions after in-
whose organdies determine the quality, quantity, and speed ternalization as a potent mitogen (67). These examples illus-
of processing. a2M complexes may be dispatched into differ- trate that latent biological factors are internalized and be-
ent routes of endocytic/lysosomal pathways, including in- come activated within the cell to mediate such diverse
tracellular sequestration, translocation from the endocytic biological responses as cell death or cell proliferation. Be-
compartment to the cytosol, transcytosis in cells with pola- cause a2M mediates uptake of peptides of diverse biological
rized functions, and degradation with or without engaging functions it is reasonable to predict that it functions, at least
the antigen processing apparatus (Fig. 2 and Fig. 3). In ad- in some instances, as a biological response modifier through
dition, ligands dissociating early in this pathway may be the above mechanism. In vivo, the ricin A chain associates
routed differently and assume biological functions different with a large molecular weight serum protein, identified as
from those ligands that remain bound to a2M. Differences a2M, and complexes remain detectable in circulationhours
in the processing of a2M complexes are inferredfrom several after administration of toxin (13). In vitro, the binding ki-
observations. Uptake of a2M complexed with Serratia mar- netics of the ricin A chain to activated a2M show a strong
cesceas proteinase, a highly cytotoxic proteinase, does not resemblance to that of IL-1 (13, 40). Complexes formed be-
mediate protection from cytotoxicity. Instead, upon inter- tween ricin A chain and Fa2M retain cytotoxicity. As ricin
nalization of the complex by fibroblasts the proteinase is A chain iscytotoxic only ifinternalized,itis concluded that
reactivated and mediates cell killing. In the absence of a2M, Fa2M closely mimics the function of the ricin B chain in
the Serratia proteinase is significantly less toxic. Therefore, mediating internalization.
the cytotoxicityof the Serratia marcescens proteinase depends
on its internalization as a complex with a2M through
a2M, MORE THAN A PROTEINASE INHIBITOR
a2MR/LRP and on its dissociation from the complex within
the cell (65). In another model, enzyme replacement in an A shift in perspective on the biological function of a2M has
a-galactosidase deficient cell line was accomplished by ac- become apparent in the recent literature. Previous reports

o
o
pee
pmteinase
Y
ry.2MR/LFi.P
A cytokine native a2M activated a2M

/4..
//

p.,

/ /

Figure 2. Proposed model of assembly of a2M-peptide-proteinase-cytokine complexes and cell targeting. The tetrameric nature of a2M
is shown (see ref 24). The stepwise assembly of a complex is shown starting with cross-linking by transglutaminase of a peptide to native
a2M (step 1). Activation of a2M (step 2) results in a distinct conformational change that traps bound peptides as well as the proteinase
that activates a2M. Activated a2M expresses cytokine and toxin binding sites and receptor binding sites on each of its four tips (step 3).
A high affinity type of interaction between a2M and a2MR/LRP as suggested in ref 96 is shown (step 4). A cell surface redox enzyme
system (Trx = thioredoxin) may release cytokines from the complex (ref 40). Released cytokines can now interact with specific receptors.
In step 5, a2M and bound peptides are internalized via cr2MR/LRP. After acidification of the endosome, the a2MR/LRP dissociates from
the ligand and recycles intact to the surface. The fate of endocytosed complexes may depend on the cell type and the differential sorting
and processing of endosomes (see Fig. 3).

3348 Vol. 6 December 1992 The FASEB Journal BORTH

 tion from the complex early in the endocytic pathway,
whereas peptides irreversibly bound are more likely to reach
late lysosomal compartments where proteolysis takes place.
endocyt os is The importance of this is best illustrated by cross-linking
2. receptor recyci ing studies of toxins to tumor surface antigen-specific antibodies,

ED
regurgitat ion
a complex termed immunotoxin (IT). Disulfide bond linkage
3. sequestration
I. peraeat ion
of toxins to Ab’s exhibits cell killing activity due to endocyto-
5. degradation
6. tronscytosis 0 sis of IT and intracellular
Thioether linkage,
which has not been demonstrated,
however,
reductive release
the intracellular
abolishes
of the toxin.
cleavage
the release
of
of
the ricin A chain and arrests the cytotoxicity of IT (57).
14 actvoted a2fl: V: o2tR/LRP; flo: peptide cro,,-iinked to cz211; More than 2 decades ago a2M was identified as a major
Its-st peptide disuifide bor,ded to u211; component of the a-globulin fraction of serum that modu-
lates cell growth. Early studies showed that a2M affected cell
Figure 3. Proposed model of possible endosomal pathways of a2M
growth in a number of different culture systems, including
complexes. Upon internalization, a2M complexes may be dis-
B cells (69), fibroblasts (70), neurite outgrowth (71), T cell
patched into different routes of endocytic/lysosomal pathways. Pas-
proliferation (72), and others (reviewed in refs. 73, 74). It
sage through the endocytic compartment and destination of ligands
was proposed that a2M exerted growth modulation on its
bound to a2M may depend on whether they are dissociable early
in the pathway or remain bound to a2M. Early dissociation of own (70) by binding mitogenic proteinases (69), releasing
ligands may allow a dissimilar routing of that of a2M and ligands small biomediators it carried (71), or acting on T cellgrowth
irreversibly bound to it. mediators (72). The cell growth modifying properties of a2M
finally led to its identification as a cytokine binding factor.
The circumstantial evidence for a2M as a modulator of cell
regarded the interaction of a2M with proteinases or the growth was already substantial when a direct link between
cleavage of the thiol ester as a mechanism of inactivating growth factors and a2M was established by demonstrating
a2M, reflecting the concept that a2M fulfills its function by that platelet-derived growth factor (PDGF) associated with
binding proteinases and that these complexes are ‘cleared” serum a2M (75). It was noted that PDGF interacted with
without further biological consequences or significance. a2M in a different way than proteinases, and a thiol-disulfide
More recent literature, however, considers the cleavage of the exchange reaction was proposed. Similarly, platelet-derived
thiol ester as a means of activating a2M, probably to reflect transforming growth factor-f3 (TGF-i3) was found to cova-
the concept that this form of a2M acquires new functions lently associate with a2M (76). Cytokines that bind to a2M
reminiscent of a binding, carrier, and targeting protein that during clotting of platelet-rich plasma retain mitogenic ac-
can modulate biological responses. As outlined previously, tivity, whereas a2M purified from platelet-poor plasma lacks
ligands can bind to a2M in different ways. Proteinases that mitogenic activity (77). a2M-cytokine complexes also
attack native a2M become instantly trapped. The carbonyl promoted cellgrowth under serum-free conditions at a2M
group of the reactive glutamate generated by the cleavage of concentrations of 3 nM (70), but only a fraction (< 1%) car-
the thiol ester can form an ester bond with a hydroxyl group ries a cytokine molecule (78). In addition, a2M inhibited the
or an amide bond with the c-amino group of accessible ly- binding of PDGF as well as of ]DF-/3 to their growth factor
sines of the proteinase, thereby mediating covalent linkage. receptor without inhibiting growth factor activity (76, 79).
Ester bond formation between activated a2M and peptides Subsequent studies showed that a2M synergistically en-
is presently unknown, but for complement components C3 hanced PDGF-stimulated proliferation of fibroblasts by
and C4 this type of linkage is critical to biological activity. twofold (78). These data combined with the previous obser-
The half-life of the reactive glutamate is short, and if lysines vations made it possible to conclude that a2M functions as
are not accessible, the proteinase may become trapped a carrier protein for some cytokines by binding these in a
without being covalently cross-linked. Intracellular acidifica- reversible manner with relatively high affinity (K 10 to
tion and/or other chemical processes, e.g., reduction, allow 109 M) (74, 80).
noncovalently trapped proteinases to escape from the com- In some cell systems, mechanisms may exist that mediate
plex (7). The extent of covalent linkage of proteinases shows dissociation of cytokines from a2M either on the cell surface
wide variation (1, 2, 7). The glutamate can also react with or in an endocytic compartment that allows the cytokine to
water or an accessible lysine of a nonproteolytic peptide that interact with the signal transduction cascade. Mechanisms
becomes accidentally entrapped. Insulin was shown to bind that could mediate dissociation of the cytokine from the com-
to a2M in this way (ii, 68). Disulfide linkages between pro- plex are endosomal/lysosomal reduction and acidification. In
teinases and a2M, as described for cytokines, have not been vitro studieshave shown that the thioredoxin system iscapa-
reported. An intracellularmechanism that releasesthe pro- ble of releasing IL-1/3 from a2M (40). On these grounds we
teinase after forming e(’y-glutamyl)lysine bonds with a2M is are proposing that in tissues, membrane-associated
not known. Therefore, activation of proteinases probably oc- thioredoxin may be involved not only in reductive activation
curs only in the case of noncovalent association with a2M, of toxins, but also in that of cytokines. Nonspecific dissocia-
but not where covalent bonds form. The irreversiblecovalent tion due to poor affinity is a less likely explanation for the bi-
capture of proteinases, however, and the receptor-mediated ological effects produced by a2M cytokine or toxin com-
endocytosis of the complexes have established the role of plexes because affinity constants indicate stable noncovalent
a2M as a ‘panproteinase inhibitor” and highly efficient or covalent complex formation. Dissociation of cytokines
scavenger that clears most proteinases by directing them to from the complex are achieved only by ‘extraction” using
intracellular compartments where complexes can be acidification, reduction, or organic solvents, all of which are
degraded. The chemistry of linkage between a2M and the methods previously used to extract proteinases from a2M
peptide is likely to determine the routing of the ligand along (7). Extraction methods were also required to ‘unmask”
the endocytic process. Cleavable or reversible linkages may cytokine epitopes in order to obtain accurate cytokine meas-
allow peptides to assume biological function after dissocia- urements by immunosorbent techniques (81). Binding of the

BIOLOGY OF a2M 3349

 a2M-cytokine complex to the cytokine receptor is also un- cations. The zinc-dependent binding of IL-1/3 requires both
likely as cytokines are sterically hindered as shown by the histidine and cysteine residues, which are known zinc bind-
failure of specific antibodies to interact with a2M-bound ing sitesand become accessible in activated a2M (40). The
cytokines or toxins (12, 13, 40). In contrast, ce2M-cytokine initial association step of a2M and IL-I/I and other cytokines
complexes retain affinityfor a2MR/LRP. The interactionof is hydrophobic in nature and is stabilized by the formation
monoclonal antibodies against the receptor binding sitein of disulfidebonds. Our studiesshowed that cytokines includ-
a2M isnot affectedif IL-Ij is complexed to a2M (12). In ad- ing IL-i/I,TGF-f31, and bFGF bound only to fullyactivated
dition, clearance of TGF-f31-a2M complexes in mice is a2M or to forms that migrated as intermediate bands be-
equivalent to that of activated a2M alone, but differs from tween S and Fa2M in native electrophoresis. These inter-
clearance of free TGF-/3 1 (82). mediate bands are seen after purification and storage of na-
Discordant reports as to whether a2M acts as an inhibitor tive a2M and represent unstable forms of partially activated
of cytokine function may originate primarily in experimental a2M in which one to three thiol esters were cleaved. Thus,
variability, which makes interpretation of results difficult. if only poor resolution is achieved by electrophoretic means,
These variablesinclude the celllineused to testbiologicalac- early Fa2M products cannot be unambiguously distin-
tivity, whether cells produce a2M and/or express guished from Sa2M. For unequivocal discrimination of a2M
a2MR/LRP, and the way cells process the complexes. The forms and itscytokine complexes, we have suggested using
source of serum used significantly influences binding ofmonoclonal antibodies that react with either native a2M or
growth factor to a2M. Human and rabbit serum were most activated ct2M (12). Using these tools, we concluded that
effectivein inhibiting binding of TGF j31 and TGF /32 to most cytokines that bind do so primarily, ifnot exclusively,
receptors (83), whereas fetal bovine serum was the least to activated forms of a2M, but exceptions cannot be ruled
effective. Differences in a2M serum concentrations of as out. Binding and equilibrium kineticsshowed some variabil-
much as 10-fold probably account for some variability. ity,but in each instance disulfide-bonded complexes formed
Qualitative differencesexistwhen complexes are generated with activated ca2M. Others have reported that some
in cell cultures or clotting of platelet-richplasma occurs cytokines bind also to native a2M and do not form disulfide
rather than when purified cytokines are added to the system bonds with activated a2M and have proposed that there
or complexes are produced by mixing the purified compo- might be several cytokine binding sites on a2M that are
nents. The complexity of this issue is reflected by studies that related or unrelated to thiol ester activation.
addressed PDGF production by different macrophage popu- The chemistry of growth factor a2M interaction has led to
lations. Concanavalin A-stimulated human alveolar and the hypothesis that complex formation could either be in-
peritoneal macrophages produce significant amounts ofhibited or promoted by other biological factors as well as by
PDGF under serum-free conditions (84). However, antibod- synthetic compounds that modify disease progress. Heparin
ies directed against PDGF neutralized the mitogenic activity and related compounds inhibit binding of TGF-/31 to a2M,
of alveolar macrophage-derived PDGF, but not that of which led some to propose that these compounds could be of
peritoneal macrophage-derived PDGF, because this as- therapeutic value in diseases such as atherosclerosis
sociated with a2M synthesized by this cell lineage, thereby promoted by heparin binding growth factors (87). Because
masking its epitopes. Complexes, however, could be partially heparins also have anticoagulant activity, attempts are under
dissociated by acid treatment and released PDGF could be way to find heparin analogs or fragments that affect growth
neutralized by antibodies. factor a2M interaction, but not the blood coagulation sys-
Physicochemical and functional data suggest that tem. Such a compound has been found in fucoidan, a sul-
methylamine-activated a2M closely resembles the structure fated polysaccharide derived from marine algae (87) . D-
and function of proteinase-activateda2M (1,2, 7). There- penicillamine (D-PA) and chrysotherapeutic drugs (gold-
fore, methylamine-activated a2M has been used frequently containing drugs) used frequently in the treatment of rheu-
to study cytokine binding. Studies with proteinase-activated matoid arthritis and other autoimmune diseases influence
a2M gave variable results.The reasons for these variabilities IL-i/I a2M complex formation. D-PA, a drug with a free
were recently addressed by studying binding of TGF-/3 I to sulfhydryl group and also used as an antidote for heavy me-
proteinase-activated a2M by varying the concentration as tal intoxication, inhibits IL-i/I a2M complex formation at
well as type of proteinase (85). This showed that the therapeutic concentrations. Because the inhibitory effect of
stoichiometry of a2M-proteinase complexes (molar ratio of D-PA could be reversed by the addition of zinc, it appeared
a2M to proteinase) as well as the proteinase type can that the inhibitory effect was due to the chelating effect
significantly determine cytokine binding to a2M-proteinase rather than the potentially reducing effect of D-PA. Gold
complexes. In in vitro experiments, cr2M is frequently satu- compounds were found to enhance complex a2M-IL-1/3 for-
rated with excess proteinases by ‘squeezing” in too many mation in the absence as well as presence of zinc. This sug-
proteinase molecules into the a2M trap, which results in ob- gested that gold acted like zinc, which mediates complex for-
struction of cytokine binding sites. In contrast, reaction of mation between IL-i/I and a2M (40, 69; W. Borth,
a2M with an equimolar amount of proteinase produces com- unpublished observations).
plexes that avidly bind cytokines. In vivo, a2M is always in The possible generation of complexes in vivo is strongly
excess over proteinases, even at sites of inflammation (86), supported by experimental data that a2M-proteinase-
and binary complexes are formed (a2M:proteinase = 1:1) cytokine complexes are produced in tissue culture and that
that favor cytokine binding. cytokines released by platelets and perhaps other cells associ-
The number of cytokines that bind to a2M is steadily in- ate with a2M in the clotting plasma (70, 75, 76, 84, 88). Acti-
creasing (reviewed in refs 73, 74). Not all cytokines bind to vation of serum by immune complexes was also shown to
a2M, and there seem to be differenceswith respect to the mediate complex formation between IL-I/I and a2M and its
binding mode. For example, IL-1/3 binds only to homologues, and this mechanism is thought to be significant
methylamine- or proteinase-activated a2M (12, 40). This in- in autoimmune disease and immune defense processes (89).
teraction requires zinc and isunlike that of nerve growth fac- Complexes may be rapidly eliminated from circulation or
tor (NGF), ‘IDF-f31, and basic fibroblast growth factor may be produced and “cleared” by tissue-fixed cells, thereby
(bFGF), which bind to activateda2M in the absence of metal making detection and measurement elusive. In unusual con-

3350 Vol. 6 December 1992 The FASEB Journal BORTH

 TABLE 1. Summary of binding mechanisms and functions of a2M

Ligand Mechanism that mediates binding Biological significance

Proteinases Trapping, e(-y-glutamyl)lysine linkage “Proteinase inhibition” clearance, targeting,

import and processing of foreign peptides
Cytokines Hydrophobic, zinc, disulfide linkage Alteration, targeting, clearance of cytokine,
modulation of cell growth and function
Toxin Disulfide linkage Unknown
Other peptides Transglutaminases catalyse Unknown
e(y-glutamyl)lysine linkage

Bacteria Unknown Opsonization

Virus Terminal sialic acids Opsonization, inhibition of virus infection

Various peptides Ion exchange Unknown

Zinc Coordinated chelation of peptides, Carrier

e.g., IL-i/l, others?

ditions, however, activated a2M may be demonstrable in This work was supported by grant no. 4162 from the Oester-
vivo, whether it is complexed with proteinases (86) and/or reichische Nationalbank. I am grateful to my colleagues P. C. Har-
with ligands bound through disulfide bonds (13). pel, L. Mayer, J. Rand, and L. Sottrup-Jensen for helpful discus-
sions and revision of the manuscript.

a2M, A PROTEINASE INHIBITOR IS SEEKING A

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