α1-Microglobulin

Bo Åkerström; Lennart Lögdberg

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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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α₁-Microglobulin

Bo Åkerström and Lennart Lögdberg.

Author Information and Affiliations

α1-Microglobulin is one of the three original members of the lipocalin superfamily. It has been found in mammals, birds, amphibians and fish and is distributed in plasma and extravascular compartments of all organs. α₁-Microglobulin has a free cysteine side-chain located in a flexible loop, giving the protein reductase and dehydrogenase properties with a broad biological substrate specificity. Three lysyl residues located around the opening of the lipocalin pocket carry yellow-brown modifications originating from the binding and degradation of heme and kynurenin, the latter a tryptophan metabolite. We have suggested that α₁-microglobulin is involved in defending tissues against oxidation by heme, kynurenin and reactive oxygen species.

Introduction

α₁-Microglobulin (α₁m) was discovered in human urine 40 years ago and was named in the tradition of plasma proteins, reflecting its small size (26 kDa) and its electrophoretic migration slightly behind albumin.¹ The protein was characterized by several research groups and given the alternative names protein HC, “human complex-forming protein, heterogeneous in charge”² and α₁-microglycoprotein.³ α₁M, retinol-binding protein (RBP) and β-lactoglobulin were the three original members when the lipocalin family was defined in 1985.⁴

The physiological role of α₁m has only recently been clarified. Immunoregulatory (mainly suppressive) properties of α₁m were identified (see below), but did not seem distinct or strong enough to constitute the major function of the molecule in vivo. However, several very recent reports have suggested that α₁m may play a biological role as an anti-oxidant with oxidant-scavenging and enzymatic reductase properties. In this review, we will describe the structural features of α₁m, its distribution among tissues and species and the anti-oxidation and immunoregulatory properties of this lipocalin.

Structure

Peptide Chain

The full sequence of human α₁m was first reported by Kaumeyer et al.⁵ The protein was found to consist of 183 amino acid residues. Since then, ten additional α₁m cDNAs and/or proteins have been detected, isolated and/or sequenced, from other mammals,⁶^-¹⁷ birds,¹⁸ amphibians, ¹⁹ and fish²⁰^-²² (Table 1). The length of the peptide chain of α₁m differs slightly among species, due mainly to variations in the C-terminus. Alignment comparisons of the different deduced amino acid sequences show that the percentage of identity varies from approximately 75-80% between rodents or ferungulates and man, down to approximately 45% between fish and mammals. A free cysteine side-chain at position 34 is conserved. This group has been shown to be involved in redox reactions, in complex formation with other plasma proteins and in binding to a yellow-brown chromophore (see below). Computerised 3D models²³ based on the known X-ray crystallographic structures of other lipocalins suggest that Cys34 is solvent exposed and located near the opening of the lipocalin pocket (fig. 1). Complement factor C8γ, another lipocalin, also carries an unpaired Cys in position 34 that is involved in the formation of the active C8 complex.²⁴ In yet another lipocalin, prostaglandin D synthase, a free Cys65 has been shown to be of importance for the catalytic activity of the enzyme.²⁵

Table 1

α₁M and bikunin are encoded by a single gene and co-expressed as a precursor-protein. This genetic construction has been found in many species.

Figure 1

Three-dimensional model of α₁m. The model was generated using Swiss-Pdb Viewer and Swiss Model by alignment of the human α₁m amino acid sequence with rat epididymal retinoic acid-binding protein (E-RABP). β-strands are shown in (more...)

Carbohydrates

Human α₁m is substituted with oligosaccharides in three positions, two sialylated complex-type, probably diantennary carbohydrates linked to Asn17 and Asn96 and one more simple oligosaccharide linked to Thr5.²⁶^-²⁸ The carbohydrate content of α₁m proteins from different species varies greatly, though, ranging from no glycosylation at all in Xenopus leavis¹⁹ over a spectrum of different glycosylation patterns. However, one glycosylation site, corresponding to Asn96 in man, is conserved in mammals, suggesting that this specific carbohydrate may be functionally important.

Chromophore

α₁M is charge- and size-heterogeneous. Tightly bound, heterogeneous, brown-coloured prosthetic groups have been proposed as responsible for the heterogeneity.²⁹ The heterogeneity and brown colour are universal properties of α₁m from all species and at least parts of the brown materials are attached intracellularly to the protein.²⁰^,³⁰ Several structurally different chromophores attached to multiple residues may explain the observed charge and size heterogeneity of the protein. Covalently linked coloured substances have been localized to Cys34,²⁹ and Lys92, Lys118 and Lys130, the latter with molecular masses between 100 and 300 Da.³¹ Molecular modelling suggests that all four residues are located at the entrance of the lipocalin cavity (fig. 1). Recently, the tryptophan metabolite kynurenine was found covalently attached to lysyl residues in α₁m from urine of hemodialysis patients and appears to be the source of the brown colour of the protein in this case.³²

Lipocalin Ligand

Available data suggest that many different ligands can be fitted into the pocket of α₁m. Several hydrophobic substances, including retinol, have been extracted from α₁m, but in molar quantities never more than approximately 1/1000 of the protein.³³ A 282 Da lipophilic substance was copurified with the peptides containing Lys92, Lys118 and Lys130.³¹ Recently, several papers demonstrated that heme binds specifically to α₁m in plasma, urine and other tissue fluids.³⁴^-³⁶ The affinity constant (K_a) was approximately 1-2 x 10⁶ M^-1, and the binding evolutionarily conserved.³⁶ A physiological role as a heme-scavenger was therefore proposed for α₁m (see below).

Synthesis

Cosynthesis with Bikunin

The gene for α₁m is called AMBP (Alpha-1-Microglobulin-Bikunin Precursor gene) because it also encodes bikunin, another plasma protein5 (fig. 2). Bikunin³⁷ is a common subunit of a group of protein/carbohydrate complexes that constitute the inter-α-inhibitor family. Its members are plasma and tissue proteins that have proteinase inhibitor activity³⁸ and serve as structural components of extracellular matrix.³⁹ Curiously, transcription of the AMBP gene, which takes place mainly in the liver, produces an mRNA that is translated into a precursor protein consisting of a 19 amino acid residue signal peptide and the α₁m and bikunin proteins connected by a linker tripeptide. The linker tripeptide and the last amino acid in α₁m, an arginine, constitute a basic cleavage site, R-V-R-R, that is recognized by furin and other subtilisin-like proprotein convertases (SPCs).⁴⁰ Before secretion, the α₁m- and bikunin-components are separated by proteolytic cleavage in the late phase of post-translational processing in the Golgi-system.⁴¹ The bikunin-part is modified by attachment of a glycosaminoglycan (chondroitin sulfate chain) to Ser10⁴²^,⁴³ and the bikunin molecules are linked to larger subunits, so-called heavy chains, via N-acetylgalactosamine residues in the glycosaminoglycan.⁴⁴^,⁴⁵ Both α₁m and bikunin then leave the hepatocyte and enter the blood. No connection has been found between α₁m and bikunin after they leave the hepatocyte and the reason for the cosynthesis of α₁m and bikunin is not understood. Moreover, it has been shown in different expression systems that both α₁m and bikunin can be expressed alone.²⁰^,⁴⁶^-⁴⁹ In spite of this, the α₁m/bikunin genetic construction is conserved in all species where α₁m has been found.

Figure 2

Synthesis of α₁m and bikunin in human liver. The α₁-microglobulin/bikunin precursor gene (AMBP), codes for the precursor-protein α₁m/bikunin. The signal peptide and the α₁m-part are encoded by exons 1-6 and the linker peptide (more...)

Gene

The AMBP gene from man⁵⁰ and mouse⁵¹ has been cloned. Of its ten exons, the first six code for α₁m (fig.2). The AMBP gene has been mapped to the 9q32-33 region in man⁵² and to chromosome 4 in mouse,⁵³ sites in both species where other lipocalin genes are clustered (reviewed in ref. 54). Intron F, which separates the α₁m-exons from the bikunin-exons, contains retroposons and other repeated structures, suggesting that it is a recombinatorial hot-spot.⁵¹ This could have provided the basis for a fusion between an ancestral lipocalin gene (α₁m) with an ancestral Kunitz inhibitor gene (bikunin).

Expression and Distribution

Liver, Blood and Kidney

Early quantitative tissue distribution studies revealed liver, blood plasma, and kidney as major sites of α₁m localization.⁵⁵ This pattern reflects the major phases of the metabolism of the protein. (1) The liver is a major site of synthesis of α₁m in adult tissues in all species studied.⁵⁶^-⁵⁹ (2) α₁m is then secreted to the blood, where the protein exists in free form as well as in a variety of high molecular weight complexes (see below). Both free α₁m and the complexed forms are very rapidly equilibrated between the intra- and extra-vascular compartments and their half-lifes in blood are 2-3 min.⁶⁰^,⁶¹ (3) Free monomeric α₁m passes relatively freely through the glomerular membranes out into the primary urine, where it is reabsorbed by the proximal tubular cells and catabolized.⁶² The binding of α₁m to the tubular cells is mediated by the multiligand receptor megalin, a member of the LDL-receptor gene family.⁶³ Megalin also binds albumin and the lipocalins retinol-bindning protein (RBP) and major urinary protein (MUP).⁶³

Expression

The liver is the predominant site of synthesis of α₁m. AMBP gene expression in liver is tightly regulated by a unique set of cis-elements and transcription factors known as hepatocyte nuclear factors (HNF 1-4).⁶⁴^-⁶⁶ Several secondary and minor locations of α₁m expression have been reported. Thus, α₁m mRNA has been detected in adult kidney,¹²^,²¹ pancreas,⁶⁷^,⁶⁸ stomach⁶ and blood cells.²¹ Traditionally, the expression of α₁m has been considered as constitutive, but lately this view been challenged. Thus, induction of α₁m-bikunin expression in kidneys by oxalate was recently demonstrated,⁶⁹ suggesting a regulatory role of α₁m in oxalate-containing kidney stone formation. Moreover, hemoglobin and pro-oxidants up-regulated the α₁m-expression in hepatoma and blood cell lines,⁷⁰ supporting the view that α₁m is involved in anti-oxidation and heme-protection (see below and fig. 3).

Figure 3

Anti-oxidant properties of α₁m. Pro-oxidants, exemplified by heme, the iron-containing prosthetic group of hemoglobin, myoglobin, cytochrome c and other heme-proteins, induce formation of free radicals and reactive oxygen species (ROS). These (more...)

Plasma

In human plasma, approximately 50% of α₁m forms a one-to-one complex with monomeric IgA by a reduction-resistant bond between the penultimate cysteine in the a-chain and Cys34 of α₁m.⁷¹^-⁷³ Approximately 7% is linked to albumin²^,⁷⁴ and 1% to prothrombin by a disulfide bond.⁷⁴ High molecular weight (HMW) forms of α₁m have been found in all species examined. In rat serum, α₁m is found covalently linked by a disulfide link to fibronectin⁷⁵ and by a reduction-resistant bond to the proteinase inhibitor α₁-inhibitor-3, a homologue of human α₂-macroglobulin.⁷⁶ α₁M complexes have also been detected in plaice serum.²⁰ Thus, the complex-forming ability of α₁m is conserved from fish to man, although the identity of the complex partners is not conserved. It is not known in what compartment any of the α₁m-complexes are formed.

Unusual forms of HMW α₁m have been found in plasma from patients with various pathologies; α₁m has a tendency to bind to mutated forms of coagulation factors that include a free Cys residue. The conserved unpaired Cys34 of α₁m is probably involved in the formation of these complexes. Thus, circulating complexes have been described between α₁m and factor IX Zutphen,⁷⁷ factor XII Tenri⁷⁸ and several protein C mutants.⁷⁹ In all mutants the unusual free, unpaired cysteine residue is located in an N-terminal Gla-domain (γ-carboxy glutamic acid-domain). The Gla-domains are believed to mediate the binding of the coagulation factors to membrane surfaces. It was shown by molecular modelling of α₁m and the protein C Gla-domain that electrostatic and hydrophobic interactions may attract the two proteins to each other and orient them in such a way that formation of a disulfide bond between the two free cysteines is favoured.²²

Determination of the α₁m concentration in human plasma or serum is complicated by the presence of complex forms of the protein (see above). Consequently, reports on normal α₁m-concentrations in human plasma/serum have varied. Several investigators have measured free α₁m and IgA-α₁m separately in normal serum.⁶⁴^,⁸⁰^-⁸² For example, DeMars et al⁸² found a mean concentration of 33 mg/L for free α₁m and 248 mg/L for IgA-α₁m, corresponding to a molar ratio of approximately 1:1 (∼1 μM of each). Without distinguishing between free and complexed α₁m, the values for “total” α₁m in rat, guinea pig and plaice serum were determined to be 9-16 mg/L, 26 mg/L and 20 mg/L, respectively.⁸^,¹²^,²⁰

Other Tissues

Besides its predominant localization in plasma and liver and kidney cells, α₁m has been identified in the perivascular connective tissue of most organs⁶⁷^,⁸³^,⁸⁴ and is especially abundant in epidermis of skin35,85 and epithelium of the gut.61,86,87 It is often colocalized with elastin and collagen⁸³^,⁸⁵ and was reported to bind to collagen in vitro.⁸⁸ A distribution of matrix-α₁m at various interfaces between the cells of the body and the external environment (blood/tissue, air/tissue, intestinal lumen/villi), as well as at the interface between maternal blood and fetal tissues in placenta,⁸⁹ is suggestive of a protective role of α₁m in vivo (see below). A similar distribution was seen in human⁹⁰ and mouse fetuses.¹⁵ In addition, fetal α₁m was seen in the cytoplasm of many epithelial cells that do not express the protein post-term.¹⁵^,⁹⁰

Anti-Oxidant Properties

Recent reports suggest that α₁m is involved in the defense against oxidative tissue damage (oxidative stress). The proposed anti-oxidant mechanisms are summarized in Figure 3. Pro-oxidants are constantly introduced to the human body via the environment (air, food, etc) but are also produced endogenously as metabolites in the normal homeostasis.⁹¹ Increased amounts of pro-oxidants are seen during inflammation, and oxidative stress is considered to be a major factor in the development of many conditions such as atherosclerosis, rheumatoid arthritis, ischemia/reperfusion injury, and diabetes. The pro-oxidants undergo reactions forming reactive oxygen species (ROS) and free radicals. These react with proteins, DNA and other molecules of human tissues by oxidation. These oxidation reactions are often harmful and may destroy the function of the target molecules (oxidative damage). Heme, the prosthetic group of heme-proteins, is a prominent example of an endogenous pro-oxidant and is harmful when released into the extracellular environment.

Thus, α₁m uses three major mechanisms to achieve anti-oxidation: (1) scavenging heme and other pro-oxidants, (2) inhibiting oxidation reactions, and (3) enzymatic reduction of harmful oxidation products. Additionally, a fourth mechanism enhance its anti-oxidant action: heme- and pro-oxidant-induced up-regulation of the synthesis of α₁m⁷⁰ (see above).

Heme-Scavenging

Both α₁m and its IgA-complex bind to the heme group.³⁴^-³⁶ The exposure of α₁m to erythrocyte membranes or purified hemoglobin leads to the binding of heme and the formation of a truncated form of the protein (t-α₁m) that lacks the C-terminal tetrapeptide LIPR and has heme-degrading properties. A pronounced yellow-brown colour was formed by incubating t-α₁m with heme, suggesting that at least some of the α₁m-chromophores may be heme degradation products.³⁴ The t-α₁m form is present in urine and thus is formed in vivo.³⁴^,⁶⁹^,⁹² In chronic venous ulcers, an inflammatory condition where free heme and iron released after hemolysis are considered to be pathogenic factors, α₁m was colocalized with heme and t-α₁m was continuously formed.³⁵ Based on these findings, a role as an extracellular heme-scavenger was proposed for α₁m. As mentioned above, the tryptophan metabolite kynurenine is attached to lysyl residues in α₁m from the urine of hemodialysis patients.³² Kynurenine metabolites are formed in tryptophan metabolism and are pro-oxidants, i.e., may induce oxidative stress.⁹³^-⁹⁵ The binding of heme and kynurenin and the transformation of these substances to chromophores may be two examples of a pro-oxidant scavenging mechanism of α₁m.

Inhibition of Oxidation and Enzymatic Reductase Activity

α₁M inhibited the heme- and ROS-induced oxidation of collagen, low-density lipoproteins (LDL), membrane lipids and whole cells.⁹⁶ α₁M also removed preformed oxidation products present on collagen and LDL. This suggests that α₁m may act as an oxidation repair factor. A possible mechanism for this may be the enzymatic reductase/dehydrogenase properties recently described for α₁m.⁹⁷ Thus, the protein was capable of reducing heme proteins, free iron and the synthetic compound nitroblue tetrazolium (NBT) using the electron donors ascorbate and NADH/NADPH as cofactors. The thiol group of Cys34 and the three lysyl residues of K92, K118 and K130 were found in the active site, suggesting that the chromophore or chromophore formation is linked to the reductase activities.

Immunoregulatory Properties

Due to its immunoregulatory properties, α₁m is categorized as an immunocalin.⁹⁸ It inhibits central events of the immune response in vitro. Thus, the antigen-induced cell division of peripheral blood lymphocytes was inhibited by α₁m.⁹⁹^,¹⁰⁰ The effects were species independent, i.e., similar effects on human cells were obtained with human, rat, rabbit or guinea pig α₁m.¹⁴ It was also shown that the antigen-induced interleukin-2 (IL-2) production by mouse T helper cell hybridomas was inhibited by human α₁m.¹⁰¹ Furthermore, inflammatory responses of blood cells were inhibited by α₁m; these included migration⁹⁸ and chemotaxis¹⁰² of neutrophil granulocytes and the production of free radicals and IL-1β by peripheral lymphocytes/monocytes.⁸⁸ Finally, at low serum concentrations, a strong direct mitogenic effect by α₁m on resting guinea pig and human lymphocytes was seen.¹⁰⁰^,¹⁰³^,¹⁰⁴ At the high serum concentrations (10-20%) used to measure the inhibition of antigen-stimulated lymphocytes, no direct stimulation of the cell division was seen. It is possible that the immunoregulatory effects of α₁m are related to its anti-oxidant and reductase activities, as ROS have been shown to be involved as (positive) factors in cell signalling during lymphocyte activation (reviewed in refs. 105, 106).

Cell Receptor

α₁M has been shown to bind to the surface of various white blood cells, including human peripheral B and T lymphocytes, human NK cells,⁶⁰ the human histiocytic cell-line U937,¹⁰⁷ mouse peripheral B and T lymphocytes,¹⁰⁴ and mouse T helper cell hybridomas.¹⁰¹ The binding is species independent, specific for α₁m, saturable and trypsin-sensitive, suggesting that a protein that recognizes a conserved part of α₁m is present on the surface of white blood cells. The α₁m-receptor on blood cells has not yet been identified, however.

Concluding Remarks

Above, we have reviewed the current knowledge about the lipocalin α₁-microglobulin, its structural features, its unusual synthesis as a diprotein, its tissue expression and distribution, and we have particularly highlighted recent elucidation of its anti-oxidant properties and how these may be employed in tissue protection.

Acknowledgements

The text has been revised by Linda Lögdberg, PhD.

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