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All the nine common cytokines in this study (including NT-3, IGF-1, CNTF and TGF-alpha) bind noncovalently, yet with different specificities and to different degrees, with both normal alpha 2-macroglobulins (alpha 2M) and monoamine-modified alpha 2M. The binding of NGF is by far the most efficient and is least affected by cationic proteins. The binding of NT-3 is slightly affected by cationic proteins but is completely blocked by NGF. The binding of TGF-alpha, TGF-beta 1, CNTF, and IL-6 is severely blocked by cationic proteins/NGF. We conclude that NGF and NT-3 appear to bind specifically in significant quantities to the same alpha 2M sites; but the other cytokines by comparison bind minimally, and primarily or entirely use nonspecific molecular interactions in their binding to alpha 2M.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein closely related to human alpha(2)-macroglobulin (alpha(2)M). It has been demonstrated that monoamine-activated forms of human and rat alpha(2)M and rat alpha(1)M can bind to TrkA and, respectively, inhibit and stimulate NGF-promoted neurite outgrowth, Trk phosphorylation, and intracellular signal transduction in PC12 cells. However, the effect of PZP on neurons is unknown, and the molecular mechanism of neuroinhibition by monoamine-activated alpha(2)M is still unclear. In this report, we show that methylamine-activated PZP (MA-PZP), like MA-alpha(2)M, inhibits in a dose-dependent way the NGF-promoted neurite extension and TrkA phosphorylation in PC12 cells. On the other hand, normal PZP (N-PZP) had little or no effect. In addition, the inhibitory effect of activated alpha-macroglobulins (alphaMs) was reversible upon its removal from the cell culture. In addition, PZP, as well as alpha(2)M, is neuroinhibitory without being directly cytotoxic. It is known that the activated alphaMs bind to the multiligand receptor termed low-density lipoprotein receptor-related protein (LRP) and that the receptor-associated protein (RAP) specifically blocks uptake of all known LRP ligands. To investigate the potential role of LRP in neuromodulation by activated PZP/alpha(2)M, the effect of RAP on the neuroinhibitory activities of these alphaMs was also studied. Data presented here show that RAP blocked the neurite- and Trk-inhibitory activities of both MA-PZP and MA-alpha(2)M, whereas RAP itself had no neuromodulatory effect. Hence, we conclude that these data suggest that the LRP receptor and its alphaM ligands may play a role in regulating Trk receptors.

Human alpha(2)-macroglobulin (alpha(2)M), pregnancy zone protein (PZP), rat alpha(1)M and acute-phase rat alpha(2)M belong to the alpha(2)M gene family of proteins, which can react covalently with nucleophilic monoamines to yield monoamine-activated (MA) macroglobulins. The MA forms of human alpha(2)M, PZP and rat alpha(2)M have been demonstrated previously to inhibit various neurotrophin-promoted neuronal activities, whereas MA-alpha(1)M is neurostimulatory and all native macroglobulins are generally inactive. The mechanism of neuromodulation is unknown, but it has been postulated that MA macroglobulins might inhibit neurons via their binding and sequestration of neurotrophins. This study employed a novel biotinylation-Western blot technique to compare the neurotrophin-binding properties of the four macroglobulins, and to correlate their binding activities with their known neuro-modulatory activities. In comparison with their respective native counterparts, human and rat MA-alpha(2)M bound slightly more NGF, but significantly less BDNF or NT-3. Native human alpha(2)M and PZP in general have no neuro-modulatory activity, but native PZP bound significantly more NGF, BDNF or NT-3 than either native alpha(2)M or MA-alpha(2)M, which is neuro-inhibitory. It is known that MA-PZP is neuro-inhibitory, but it fails to bind more NGF, BDNF, or NT-3 than native PZP. MA-alpha(1)M is the only macroglobulin known to stimulate NGF-promoted neurite outgrowth, but it bound NGF with similar affinities as native alpha(1)M and rat alpha(2)M; in addition, it bound significantly less BDNF or NT-3 than native alpha(1)M. All the bindings were non-covalent and appeared specific. In conclusion, PZP and rat macroglobulins are versatile carriers of neurotrophins with diverse binding capacities, and the neurotrophin-binding property does not appear to mediate the neuro-modulatory activity of these human and rat macroglobulins.

Monoamine-activated human alpha2-macroglobulin (alpha2M) has been previously demonstrated to inhibit TrkA-, TrkB-, and TrkC-mediated signal transduction. Rat alpha1-macroglobulin (alpha1M) and alpha2M are structural homologues of human alpha2M, but rat alpha1M is distinctly different from rat alpha2M in many ways and its role in the mammalian nervous system is unknown. In this report, monoamine-activated rat alpha1M was demonstrated to enhance in a dose-dependent manner nerve growth factor (NGF)-promoted neurite outgrowth in pheochromocytoma PC12 cells. Monoamine-activated alpha1M by itself, however, was neither neurotrophic nor mitogenic to PC12 cells. To investigate further its possible mode of action, the ability of monoamine-activated alpha1M and normal alpha1M to bind and to activate the NGF receptor (TrkA) was investigated. Monoamine-activated alpha1M formed a more stable complex with TrkA than normal alpha1 M, but the binding of monoamine-activated alpha1M to TrkA was adversely affected by prior stimulation of TrkA with NGF. In addition, monoamine-activated alpha1M enhanced the NGF-promoted TrkA phosphorylation and up-regulated the expression of NGF-inducible immediate-early genes (c-jun and NGFI-A) and delayed-response genes (SCG10 and transin) in PC12 cells; normal alpha1M, in contrast, produced little or no effect. This study demonstrates that alpha1M, the constitutive form of alpha-macroglobulin in the rat, possesses the ability to promote NGF-mediated differentiation in PC12 cells, possibly via its direct action on TrkA receptors and TrkA-mediated signal transduction and gene expression.

Monoamine-activated alpha2-macroglobulin (alpha2M) was shown to reduce the dopamine concentration in corpus striatum of adult rat brains and inhibit other neuronal functions in vivo and in vitro. As brain-derived neurotrophic factor, neurotrophin-4, and neurotrophin-3 are important neurotrophic factors for dopaminergic neurons, the effect of monoamine-activated alpha2M on signal transduction by trkB and trkC was investigated. The results show that monoamine-activated alpha2M binds to trkB and inhibits brain-derived neurotrophic factor/neurotrophin-4-promoted autophosphorylation of trkB in a dose-dependent manner in both trkB-expressing NIH3T3 (NIH3T3-trkB) and human neuroblastoma SH-SY5Y cells. Monoamine-activated alpha2M also blocks tyrosine phosphorylation of phospholipase C-gamma1 and extracellular signal-regulated protein kinase (ERK)-1, which are key intracellular proteins involved in trkB signal transduction. Similarly, monoamine-activated alpha2M inhibits tyrosine phosphorylation of neurotrophin-3-induced trkC and its signal transduction in a dose-dependent manner in NIH3T3 cells expressing trkC (NIH3T3-trkC). In contrast to monoamine-activated alpha2M, normal alpha2M has little or no significant inhibitory effect on the phosphorylation of trkB and trkC. In addition, the retinoic acid-promoted tyrosine phosphorylation of phospholipase C-gamma1, ERK-1, and/or ERK-2 in SH-SY5Y cells was unaffected by monoamine-activated alpha2M; this suggests that the inhibitory effect of activated alpha2M on the neurotrophin-stimulated phosphorylation of intracellular signalling proteins may be specific. Taken together, the data indicate that activated alpha2M is a pan-trk inhibitor, which by virtue of its binding to trk receptors may block trk-mediated signal transduction in dopaminergic neurons and lead to reduction of dopamine concentration in corpus striatum.

Monoamine-activated alpha-2-macroglobulin (alpha 2M) has been shown to decrease the dopamine concentrations in rat caudate putamen (CP) in vivo as well as inhibit choline acetyltransferase activities in the culture of basal forebrain neurons. In this study, we further investigated the effects of methylamine-activated alpha 2M (MA-alpha 2M) upon striatal dopaminergic function by determining whether a direct infusion of this glycoprotein will alter dopamine (DA) release in vitro from superfused CP tissue fragments. In experiment 1, an infusion of 2.8 microM MA-alpha 2M produced a statistically significant increase in DA release compared with control superfusions. In experiment 2, varying doses (0, 0.7, 1.4, 2.8, 4.1 microM) of MA-alpha 2M were tested for their capacity to alter DA release. Only the 2.8 microM dose of MA-alpha 2M was effective in producing a significant increase of DA release. In experiment 3, the normal form of alpha 2M (N-alpha 2M) at 2.8 microM was compared with the control superfusions. The infusion of N-alpha 2M produced an increase in DA release which was substantially lower than the DA increase induced by MA-alpha 2M, and not significantly different from that of the control superfusion. These results show that MA-alpha 2M, like some other neurotoxins, can markedly alter CP dopaminergic function as indicated by the acute increase in DA release following infusion of this glycoprotein, and these effects are exerted at a relatively narrow range of doses. Taken together, these data suggest that this glycoprotein, if allowed to accumulate in the central nervous system (CNS), may promote some neurodegenerative changes that can occur in disorders like Parkinson's disease.

Monoamine-activated alpha 2-macroglobulin (alpha 2M) has been shown to inhibit choline acetyltransferase in basal forebrain neurons as well as neurotrophin-dependent neuronal functions. The objective of this study was to determine whether monoamine-activated alpha 2M can affect the caudate putamen (CP) dopaminergic system in vivo. Male rats received intracranial infusions of methylamine-activated alpha 2M (0.6 nmole) and contralateral infusions of its vehicle, phosphate-buffered saline (PBS). Five days following infusion, the animals were killed, the CP dissected into three rostral-caudal segments, and assayed for dopamine (DA) using a high-performance liquid chromatography system. Within the two rostral CP segments (the approximate site of cannula placement), statistically significant (26%) reductions of DA concentrations were obtained on the alpha 2M-infused side of the CP with 90-100% of the animals showing decreases. At a more distal (caudal) site of the CP, DA concentrations showed only an insignificant (12%) reduction. No differences in DA concentrations between sides infused with bovine serum albumin versus PBS or from olfactory tubercle samples were obtained in these animals. These results demonstrate that monoamine-activated alpha 2M is capable of producing significant degeneration of the nigrostriatal dopaminergic system in vivo and suggest that this factor may play a role in age-related neurodegenerative disorders such as Parkinson's disease.

Methylamine-modified alpha-2-macroglobulin (MA-alpha 2M) has been recently shown to inhibit the biological activity of beta-nerve growth factor (NGF) in promoting neurite outgrowth by embryonic dorsal root ganglia in culture (Koo PH, Liebl DJ, J Neurosci Res 31:678-692, 1992). The objectives of this study are to determine whether alpha 2M can also be modified by larger aromatic biogenic amines such as 5-hydroxytryptamine (5HT; serotonin), the nature of interaction between NGF and 5HT-modified alpha-2-M (5HT-alpha 2M), and the effect of 5HT-alpha 2M on the neurite extension and the growth of embryonic sensory and cholinergic neurons in 2 disparate animal species (chicken and rats). This study demonstrates that each mole of alpha 2M can combine with 15.2 +/- 1.8 moles of 5HT, in which up to 4.5 +/- 0.4 moles may be covalently bonded. As determined by gel filtration and polyacrylamide gel electrophoresis studies, both 5HT-alpha 2M and normal alpha 2M combine noncovalently with NGF, but 5HT-alpha 2M by comparison can combine with NGF somewhat more effectively. In contrast to normal alpha 2M, 5HT-alpha 2M at concentrations greater than about 0.17 microM exerts a dose-dependent inhibition on the NGF-stimulated neurite outgrowth by embryonic dorsal root ganglia and dissociated cells in culture, and the inhibitory effect can be overcome by higher NGF concentrations. Both 5HT-alpha 2M and MA-alpha 2M at 1.0 microM inhibit neurite extension by embryonic rat cerebral cortical cells and seriously damage these cells in culture. Such neurite-inhibitory activity, however, can only be partially blocked by extraneously added NGF alone. Normal alpha 2M (at 1.0 microM) and 5HT (at 188 microM), on the other hand, under the identical conditions produce very little or no effect on the normal cellular and axonal growth of these cells. We conclude that alpha 2M can potentially interact with nucleophilic monoamines, including neurotransmitters, to form inhibitory complexes which may inhibit/regulate NGF-promoted neurite outgrowth and neuronal survival. In addition, higher concentrations of such complexes can seriously damage certain CNS neurons which do not depend solely on NGF for survival.

alpha 2-Macroglobulin (alpha 2M) is a rather ubiquitous protein in extracellular spaces of mammals. It is an inhibitor of endopeptidases, can be modified by aliphatic amines, and combines with a number of hormones/cytokines such as beta-nerve growth factor (NGF) [Koo PH, Stach RW (1989): J Neurosci Res 22:247]. The objective of this study is to compare the NGF-binding properties of methylamine-modified human alpha 2M (MA-alpha 2M) versus normal alpha 2M and their effects on the biological activity of NGF and neurite extension by embryonic chicken dorsal root ganglia. As determined by gel filtration, polyacrylamide gel electrophoresis, and equilibrium binding studies, these two forms of alpha 2M are similar in their binding affinities, with MA-alpha 2M binding about twice as much NGF as normal alpha 2M. Both normal alpha 2M and MA-alpha 2M combine noncovalently with NGF, and prior modification of alpha 2M is unnecessary for the binding to occur. In contrast to normal alpha 2M, MA-alpha 2M potently inhibits the biological activity of NGF and exerts a dose-dependent inhibition on the NGF-stimulated neurite outgrowth by embryonic chicken dorsal root ganglia in culture. The inhibitory effect of MA-alpha 2M can be overcome by higher NGF concentrations, but is irreversible at lower NGF concentrations. Trypsin-modified alpha 2M combines covalently and noncovalently with more NGF than normal alpha 2M but has very little neurite inhibitory activity. The mechanism of inhibition by MA-alpha 2M is discussed.

The murine nerve growth factor, when injected i.v. or, combined in vitro with plasma, was found largely associated with the mouse alpha-macroglobulin (a homologue of human alpha 2-macroglobulin). The nerve growth factor-alpha-macroglobulin complex produced is sufficiently stable to resist separation by gel filtration in 1.0 M sodium chloride, polyacrylamide gel electrophoresis, and immunoprecipitation by antibodies against alpha-macroglobulin. As determined by equilibrium binding studies and computer generated Scatchard analysis, alpha-macroglobulin apparently possesses two types of binding sites with the apparent dissociation constants of 1.2 x 10(-6) and 2.9 x 10(-9) M, respectively, saturable by 3.7 and 0.03 moles of nerve growth factor. Hence, about one mole of nerve growth factor is bound to each of the four subunits of alpha-macroglobulin. Nerve growth factor can be readily dissociated from alpha-macroglobulin in sodium dodecyl sulfate gel electrophoresis in the absence of a reductant. Procedures that affect the proteinase-binding or methylamine- activities of alpha-macroglobulin do not affect the binding of nerve growth factor, and the binding is unaffected by the presence of zinc ions or EDTA. Hence, nerve growth factor is noncovalently associated with alpha-macroglobulin at a site separate from that of the proteinase-, methylamine-, and zinc-binding sites of alpha-macroglobulin. Mouse alpha-macroglobulin can protect the nerve growth factor from inactivation by trypsin. Even in the presence of trypsin, alpha-macroglobulin-nerve growth factor complexes still can stimulate the neurite outgrowth by dorsal root ganglia of 9-day-old chicken embryos. Since alpha-macroglobulin can specifically and noncovalently carry nerve growth factor, one important role of this alpha-macroglobulin in the circulation and extracellular spaces may be to protect the nerve growth factor from proteinase inactivation.

Human α 2 -macroglobulin ( α 2 M) was established here as a major serum factor which inhibits the DNA synthesis of a mouse ovarian tumor cell line in culture. This inhibitory activity was species non-specific, dose-dependent and unaffected by serum concentrations in culture. α 2 M was cytotoxic to both murine and human tumor cell lines in culture, as determined by 2 different viability staining techniques, morphological observation and long-term in vitro culture. This report implicates α 2 M or an α 2 M-associated substance as a major cytotoxic serum factor that may be involved in endogenous cancer control processes in mammalian species.

Mouse alpha-macroglobulin (M-AMG) is believed to be a functional homologue of human alpha 2-macroglobulin (h-alpha 2M). The subunit composition, the tryptic cleavage pattern before and after methylamine incorporation and the two-dimensional tryptic-peptide mapping, however, indicate that these two proteins are structurally distinct. M-AMG is composed of two major types of polypeptides (Mr 163,000 and 35,000) together with a minor polypeptide (Mr 185,000), whereas h-alpha 2M has only one type of polypeptide (Mr 185,000). After incorporation of methylamine, there is no change in the normal tryptic-cleavage pattern of M-AMG; however, tryptic cleavage of h-alpha 2M is severely retarded [Hudson & Koo (1982) Biochim. Biophys. Acta 704, 290-303]. The N-terminal sequence of the 163,000-Mr polypeptide of M-AMG shows sequence homology with the N-terminal sequence of h-alpha 2M. The amino acid compositions of M-AMG and its two major polypeptide chains are compared. Thermal fragmentation studies show that the 163,000-Mr polypeptide is broken down into 125,000-Mr and 29,000-Mr fragments. Trypsin-binding studies show that M-AMG can bind two molecules of trypsin/molecule. Inactivations of the trypsin-binding property of M-AMG and h-alpha 2M with methylamine show similar kinetics of inhibition at 4 degrees C. A structural model of M-AMG is proposed, based on accumulated data.