Effect of Short PSG Peptides on Inflammatory Markers in Allogeneic Bone Marrow Cell Transplantation in Wistar Rats.

Short linear peptide fragments of placental trophoblastic ß1-glycoprotein (PSG) (YECE, YQCE, YVCS, and YACS) were studied in the context of their immunomodulatory effects at the level of inflammatory markers. The original host-versus-graft model was used in male Wistar rats without prior conditioning of recipient bone marrow. A composition of PSG peptide fragments was injected to animals after allogeneic transplantation of bone marrow cells in a dynamic experiment, inflammatory markers α1-acid glycoprotein (AGP, orosomucoid), α2-macroglobulin (α2M) were assayed by ELISA, and biochemical parameters (total protein, glucose, creatinine, and urea) were measured. The levels of α2M and AGP increased in response to allotransplantation, whereas administration of PSG peptides normalized serum α2M levels by the end of the experiment. The decrease in α2M level coincided with the independent effect of PSG peptide administration. The levels of total protein, glucose, creatinine, and urea in rat serum after allotransplantation were reduced throughout the experiment. Administration of PSG peptides contributed to normalization of serum total protein, creatinine, and urea levels by the end of the experiment. Administration of PSG peptides after allogeneic transplantation of bone marrow suspension contributed to normalization of the levels of α2M, total protein, creatinine, and urea, which can be interpreted as an anti-inflammatory effect of these peptides.

Substituting the Thiol Ester of Human A2M or C3 with a Disulfide Produces Native Proteins with Altered Proteolysis-Induced Conformational Changes.

Most proteins in the α-macroglobulin (αM) superfamily contain reactive thiol esters that are required for their biological function. Here, we have characterized the human α2-macroglobulin (A2M) and complement component C3 mutants A2M Q975C and C3 Q1013C, which replace the CGEQ thiol ester motifs of the original proteins with the disulfide-forming sequence CGEC. Mass spectrometry showed that the intended disulfide was formed in both proteins. The correct folding and native conformation of A2M Q975C were shown by its assembly to a tetramer, an initially slow electrophoretic mobility with a demonstrable conformational collapse induced by proteolysis, functional protease trapping, and conformation-dependent interactions with low-density lipoprotein receptor-related protein 1. However, A2M Q975C had a decreased capacity to inhibit trypsin and was more susceptible to cleavage by trypsin or thermolysin when compared to wild-type A2M. C3 Q1013C also folded correctly and was initially in a native conformation, as demonstrated by its cation exchange elution profile, electrophoretic mobility, and interaction with complement factor B, although it assumed a conformation that was distinct from native C3, C3b, or C3(H2O) when cleaved by trypsin. These results demonstrate that disulfides can substitute thiol esters and maintain the native conformations of A2M and C3. Additionally, they indicate that proteolysis is not the sole factor in the conformational changes of A2M and C3 and that thiol ester lysis also plays a role.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Exploring the interaction of anti-androgen drug-bicalutamide with human alpha-2-macroglobulin: A biophysical investigation.

Bicalutamide (BCT), a drug used in the treatment of prostate cancer, antagonises the actions of androgens, at the receptor level, thereby inhibiting the growth of prostate tumours. Alpha-2-macroglobulin (α2M), a pan-proteinase inhibitor, inhibits proteinase, regardless of specificity and catalytic mechanism. α2M is deficient in patients of advanced prostate cancer with bone metastases. Our studies explored the interaction of BCT with α2M and analysed the BCT induced structural alteration to the α2M. The result suggests that BCT decreases the antiproteolytic potential and causes structural and functional change in human α2M. UV-visible absorption spectroscopy confirms the formation of α2M-BCT complex. Fluorescence analysis shows significant quenching in fluorescence intensity of α2M upon binding with BCT. Synchronous fluorescence result suggests the interaction of BCT with α2M changed the microenvironment around tyrosine residues. Secondary structure of α2M also undergoes a slight change upon complexation with the drug as evident by shift in negative ellipticity in far UV CD spectroscopy. FTIR results confirm the alteration in secondary structure of α2M upon drug interaction. Molecular docking studies show that BCT bind to a monomer of α2M primarily through hydrophobic force. Thermodynamics parameters were determined by isothermal titration calorimetry found that the binding was exothermic in nature.

Mutations in CPAMD8 Cause a Unique Form of Autosomal-Recessive Anterior Segment Dysgenesis.

Anterior segment dysgeneses (ASDs) comprise a spectrum of developmental disorders affecting the anterior segment of the eye. Here, we describe three unrelated families affected by a previously unclassified form of ASD. Shared ocular manifestations include bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts. Whole-exome sequencing and targeted Sanger sequencing identified mutations in CPAMD8 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8) as the cause of recessive ASD in all three families. A homozygous missense mutation in the evolutionarily conserved alpha-2-macroglobulin (A2M) domain of CPAMD8, c.4351T>C (p. Ser1451Pro), was identified in family 1. In family 2, compound heterozygous frameshift, c.2352_2353insC (p.Arg785Glnfs∗23), and splice-site, c.4549-1G>A, mutations were identified. Two affected siblings in the third family were compound heterozygous for splice-site mutations c.700+1G>T and c.4002+1G>A. CPAMD8 splice-site mutations caused aberrant pre-mRNA splicing in vivo or in vitro. Intriguingly, our phylogenetic analysis revealed rodent lineage-specific CPAMD8 deletion, precluding a developmental expression study in mice. We therefore investigated the spatiotemporal expression of CPAMD8 in the developing human eye. RT-PCR and in situ hybridization revealed CPAMD8 expression in the lens, iris, cornea, and retina early in development, including strong expression in the distal tips of the retinal neuroepithelium that form the iris and ciliary body, thus correlating CPAMD8 expression with the affected tissues. Our study delineates a unique form of recessive ASD and defines a role for CPAMD8, a protein of unknown function, in anterior segment development, implying another pathway for the pathogenicity of ASD.

Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma.

As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource.

Hypochlorite-induced structural modifications enhance the chaperone activity of human α2-macroglobulin.

Hypochlorite, an oxidant generated in vivo by the innate immune system, kills invading pathogens largely by inducing the misfolding of microbial proteins. Concomitantly, the nonspecific activity of hypochlorite also damages host proteins, and the accumulation of damaged (misfolded) proteins is implicated in the pathology of a variety of debilitating human disorders (e.g., Alzheimer's disease, atherosclerosis, and arthritis). It is well-known that cells respond to oxidative stress by up-regulating proteostasis machinery, but the direct activation of mammalian chaperones by hypochlorite has not, to our knowledge, been previously reported. In this study, we show that hypochlorite-induced modifications of human α2-macroglobulin (α2M) markedly increase its chaperone activity by generating species, particularly dimers formed by dissociation of the native tetramer, which have enhanced surface hydrophobicity. Moreover, dimeric α2M is generated in whole-blood plasma in the presence of physiologically relevant amounts of hypochlorite. The chaperone activity of hypochlorite-modified α2M involves the formation of stable soluble complexes with misfolded client proteins, including heat-denatured enzymes, oxidized fibrinogen, oxidized LDL, and native or oxidized amyloid ß-peptide (Aß1-42). Here, we show that hypochlorite-modified α2M delivers its misfolded cargo to lipoprotein receptors on macrophages and reduces Aß1-42 neurotoxicity. Our results support the conclusion that α2M is a specialized chaperone that prevents the extracellular accumulation of misfolded and potentially pathogenic proteins, particularly during innate immune system activity.

Label-free relative quantification of alpha-2-macroglobulin site-specific core-fucosylation in pancreatic cancer by LC-MS/MS.

We describe a label-free relative quantification LC-MS/MS method for core-fucosylation in alpha-2-macroglobulin (A2MG) immunoprecipitated from human sera. The method utilizes endoglycosidase F partial deglycosylation to reduce glycosylation microheterogeneity, while retaining the innermost N-acetylglucosamine (GlcNAc) and core fucose. Precursor ion peak areas of partially deglycosylated peptides were obtained and site-specific core-fucosylation ratios based on the peak areas of core-fucosylated and nonfucosylated counterparts were calculated and evaluated for assay development. This assay was applied in a preliminary study of sera samples from normal controls and patients with pancreatic diseases, including pancreatic cancer and chronic pancreatitis. A2MG fucosylation levels at sites N396 and N1424 were found to decrease in both chronic pancreatitis and pancreatic cancer compared to normal controls. The two sites were identified by two peptides and their core-fucosylation ratios were found to be internally consistent. This method provides a platform to quantify fucosylation levels and can be used to study site-specific core-fucosylation aberrations in other glycoproteins for other diseases.

The monomeric receptor binding domain of tetrameric α2-macroglobulin binds to cell surface GRP78 triggering equivalent activation of signaling cascades.

α2-Macroglobulin (α2M) is a broad spectrum proteinase inhibitor that when activated by proteinases (α2M*) undergoes a major conformational change exposing receptor recognition sites in each of its four subunits. These complexes bind to two distinct receptors, namely, the low-density lipoprotein receptor-related protein (LRP) and cell surface glucose-regulated protein [Mr ∼ 78000 (GRP78)]. The latter is a very high affinity receptor (Kd = 50-100 pM) whose ligation triggers pro-proliferative and anti-apoptotic signaling cascades. Despite its four binding sites, Scatchard analysis of binding of α2M* to cells does not yield a cooperative plot. We, therefore, hypothesize that a monomeric cloned and expressed α2M receptor binding domain (RBD) should trigger comparable signaling events. Indeed, RBD or its K1370A mutant that binds to GRP78 but cannot bind to LRP regulates DNA and protein synthesis by human prostate cancer cells in a manner comparable to that of α2M*. Akt and mTORC1 activation and signaling are also comparably upregulated by α2M*, RBD, or mutant K1370A. Antibodies directed against the carboxyl-terminal domain of GRP78 are antagonists that block α2M*-mediated effects on pro-proliferative and anti-apoptotic signaling cascades and protein and DNA synthesis. The effects of RBD and its mutant were similarly blocked by these antibodies. Finally, proteolysis of α2M at pH values from 5.7 to 7.0 causes production of free RBD and RBD-containing fragments. Thus, while α2M* ligates only one GRP78 receptor molecule per α2M*, it may potentially serve as a reservoir for release of up to four binding fragments per molecule.

Multi-peptide nLC-PC-IDMS-SRM-based assay for the quantification of biomarkers in the chicken ovarian cancer model.

A novel form of ovomacroglobulin/ovostatin (OVOS2) predicted from EST data was previously identified in the chicken ovarian cancer model using a mass spectrometry-based shotgun label-free proteomics strategy. The quantitative label-free data from plasma showed a significant increase over time with the spontaneous onset and progression of ovarian cancer making it a potential protein biomarker for further study. Two other proteins of interest identified from this initial study included vitellogenin-1 (Vit-1), a lipid-transport protein tied to egg production, and transthyretin (TTR), a retinol binding transport protein currently used in the clinical management of ovarian cancer. A multiplexed protein cleavage isotope dilution mass spectrometry (PC-IDMS) assay was developed to quantify OVOS2, Vit-1, and TTR by selected reaction monitoring (SRM). A total of 6 stable isotope labeled (SIL) peptide standards were used in the assay with three tryptic peptides from OVOS2, one for Vit-1, and two for TTR. The assay was developed for use with un-depleted raw plasma combined with the filter assisted sample preparation (FASP) method and its use was also demonstrated for matched ovary tissue samples. The PC-IDMS data for the two TTR peptides did not correlate with each other with more than a 10-fold difference in concentration for all 5 time points measured. The PC-IDMS data from the longitudinal plasma samples correlated well for OVOS2 and Vit-1 whereas TTR was inconclusive. Interestingly, the absolute amount for one of the OVOS2 SIL peptides was 2-fold less compared with the other two SIL peptides. These data illustrate the successes and challenges of qualifying quantitative levels of proteins from an in-gel digestion sample preparation followed by LC-MS/MS (GeLC) label-free discovery-based approach to a targeted SRM-based quantitative assay in plasma and tissues.

Protease-activated alpha-2-macroglobulin can inhibit amyloid formation via two distinct mechanisms.

α(2)-Macroglobulin (α(2)M) is an extracellular chaperone that inhibits amorphous and fibrillar protein aggregation. The reaction of α(2)M with proteases results in an 'activated' conformation, where the proteases become covalently-linked within the interior of a cage-like structure formed by α(2)M. This study investigates, the effect of activation on the ability of α(2)M to inhibit amyloid formation by Aß(1-42) and I59T human lysozyme and shows that protease-activated α(2)M can act via two distinct mechanisms: (i) by trapping proteases that remain able to degrade polypeptide chains and (ii) by a chaperone action that prevents misfolded clients from continuing along the amyloid forming pathway.

Differential expression of alpha 2 macroglobulin in response to dietylstilbestrol and in ovarian carcinomas in chickens.

BACKGROUND: Alpha 2 macroglobulin (A2M; also known as ovostatin), a homotetrameric protein with four disulfide-linked subunits, has the unique feature of inactivating/inhibiting most known proteases including serine-, threonine-, cysteine-, aspartic- and metalloproteases. In chickens, A2M has been identified and characterized biochemically, but little is known of its functional role(s) in the oviduct, hormonal regulation of expression or its expression in ovarian carcinomas in chickens. Therefore, we investigated estrogen regulation of A2M gene expression during development of the chicken oviduct, and its expression in normal and cancerous ovaries from chickens. METHODS: To determine tissue-specific expression of A2M in chickens, we collected various organs from male and female chickens and performed RT-PCR analyses. To examine A2M gene expression in the oviduct of 1-week-old female chicks that received a subcutaneous implant of 15 mg DES in the abdominal region for 20 days, we performed RT-PCR, qPCR and in situ hybridization analyses using cDNAs from control- (n=5) and DES-treated oviducts (n=5), and then each segment of the oviduct from DES-treated chicks. To determine if A2M is a biomarker of ovarian cancer in hens, we collected cancerous (n=10) ovaries from a total of 136 chickens which had completely stopped egg-laying and performed RT-PCR and in situ hybridization analyses. RESULTS: We found that A2M is most abundant in the chicken oviduct, specifically luminal (LE) and glandular epithelia (GE), but it was not detected in any other tissues of either sex. We then determined that DES (dietylstilbestrol, a synthetic nonsteroidal estrogen) increased A2M mRNA only in LE and GE of the oviduct of chicks. Further, expression of A2M was most abundant in GE of endometrioid adenocarcinoma of cancerous, but not normal ovaries of hens. CONCLUSIONS: Collectively, results of the present study indicate that A2M is novel estrogen-stimulated gene expressed in LE and GE of the chicken oviduct and may be used for monitoring effects of therapies for ovarian cancer in laying hens.

Modification of mouse A2M B (620-792) and A2M N (168-230) by malondialdehyde and acetaldehyde attenuates the proteinase and TGF-ß1 binding ability of A2MB.

Acetaldehyde and malondialdehyde react covalently with cellular proteins forming protein-malondialdehyde-acetaldehyde adducts thus modulating their biochemical functions. Alpha-2 macroglobulin, an acute phase protein produced by liver binds to cytokines, growth factors and neutralizes proteinases. In this study we examined the formation of MAA adducts of N-terminal and bait region of mouse A2M and their effect on modulating its proteinase and TGF-ß1 binding activities. Adduct formation abrogated the binding of bait region with TGF-ß1, trypsin, and elastase. TGF-ß1 induced NO production was also suppressed. Acetaldehyde and MDA adduction of A2M may have physiological consequences in alcoholic patients.

The protease inhibitor alpha-2-macroglobulin-like-1 is the p170 antigen recognized by paraneoplastic pemphigus autoantibodies in human.

BACKGROUND: Paraneoplastic pemphigus (PNP) is a devastating autoimmune blistering disease, involving mucocutaneous and internal organs, and associated with underlying neoplasms. PNP is characterized by the production of autoantibodies targeting proteins of the plakin and cadherin families involved in maintenance of cell architecture and tissue cohesion. Nevertheless, the identity of an antigen of Mr 170,000 (p170), thought to be critical in PNP pathogenesis, has remained unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using an immunoprecipitation and mass spectrometry based approach, we identified p170 as alpha-2-macroglobuline-like-1, a broad range protease inhibitor expressed in stratified epithelia and other tissues damaged in the PNP disease course. We demonstrate that 10 PNP sera recognize alpha-2-macroglobuline-like-1 (A2ML1), while none of the control sera obtained from patients with bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus and normal subjects does. CONCLUSIONS/SIGNIFICANCE: Our study unravels a broad range protease inhibitor as a new class of target antigens in a paraneoplastic autoimmune multiorgan syndrome and opens a new challenging investigation avenue for a better understanding of PNP pathogenesis.

A chemical approach to immunoprotein engineering: chemoselective functionalization of thioester proteins in their native state.

Less than 6 feet under: Serum proteins C3, C4, and alpha(2)M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.

Human alpha2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3.

Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.

Incorporation of low molecular weight molecules into alpha(2)-macroglobulin by nucleophilic exchange.

alpha(2)-Macroglobulin (alpha(2)M) is a proteinase inhibitor that functions by a trapping mechanism which has been exploited such that the receptor-recognized, activated form (alpha(2)M( *)) can be employed to target antigens to antigen-presenting cells. Another potential use of alpha(2)M( *) is as a drug delivery system. In this study we demonstrate that guanosine triphosphate, labeled with Texas red (GTP-TR) formed complexes with alpha(2)M( *) following activation by proteolytic or non-proteolytic reactions. Optimal incorporation occurred with 20 microM GTP-TR, pH 8.0 for 5h at 50 degrees C. NaCl concentration (100 or 200 mM) had little effect on incorporation at this pH or temperature, but was significant at sub-optimum temperature and pH values. Maximum incorporation was 1.2 mol GTP-TR/mol alpha(2)M( *). PAGE showed that 70-90% of the GTP-TR is bound in a SDS/2-mercaptoethanol resistant manner. Guanosine, adenosine, and imidazole competed with GTP-TR to form complexes with alpha(2)M( *).

Molecular cloning and characterisation of a thioester-containing alpha2-macroglobulin (alpha2-M) from the haemocytes of mud crab Scylla serrata.

Molecular approaches were used to clone thioester-containing alpha2-macroglobulin (alpha2-M) genes in the haemocytes of mud crab Scylla serrata. The full length sequence of alpha2-M was determined by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA clone consists of 5491bp with an open reading frame (ORF) of 4986bp encoding a protein of 1662 amino acids with 22 residues signal sequence. The calculated molecular mass of the mature protein is 184.2kDa with an estimated pI of 8.41. The S. serrata alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of S. serrata has an overall similarity of 68% and 48% to that of kuruma shrimp Marsupenaeus japonicus and American horseshoe crab Limulus polyphemus, respectively. Phylogentic analysis revealed that S. serrata alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of S. serrata increased significantly in 24h- and 48h-post lipopolysaccharide (LPS) injection.

Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (alpha2-M) from the haemocytes of tiger shrimp Penaeus monodon.

An alpha 2-macroglobulin (alpha2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5'-untranslated region, and a 327 bp 3'-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7 kDa with an estimated pI of 5.30. The P. monodon alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species alpha2-M showed that the overall structure is evolutionarily conserved and phylogenetic analysis revealed that P. monodon alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48 h post-peptidoglycan (PG) injection, but returned to the original values in 72 h post-PG injection.

Limited mutations in full-length tetrameric human alpha2-macroglobulin abrogate binding of platelet-derived growth factor-BB and transforming growth factor-beta1.

alpha2-Macroglobulin (alpha2M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), and carries beta-amyloid peptide. alpha2M may also trigger cell signaling by binding to the low density lipoprotein receptor-related protein (LRP-1) and/or other cell surface receptors. Based on studies with recombinant alpha2M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the alpha2M subunit. However, because intact alpha2M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the alpha2M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length alpha2M. These mutations did not perturb the tetrameric structure of alpha2M, reaction with proteases, the thiol ester bonds, or binding to LRP-1. A single mutation (E730R) completely blocked binding of platelet-derived growth factor-BB to intact alpha2M. E730R did not alter TGF-beta1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-beta1 binding associated with alpha2M conformational change. These studies demonstrate that growth factor binding to intact alpha2M is specific, involving a defined region of the alpha2M subunit. The exact sequences required for binding different growth factors may be non-identical, mimicking the model of the bait region in which different proteases target adjacent and sometimes overlapping sequences.