Inhibition of Human Pepsin and Gastricsin by α2-Macroglobulin

The inhibitory effects of human α₂-macroglobulin (α₂-M), a major plasma proteinase inhibitor, on human pepsin and gastricsin were investigated. The activities of pepsin and gastricsin towards a protein substrate (reduced and carboxymethylated ribonuclease A) were significantly inhibited by α₂-M at pH 5.5, whereas those towards a peptide substrate (oxidized insulin B-chain) were scarcely inhibited. Under these conditions at pH 5.5, pepsin and gastricsin cleaved α₂-M mainly at the His⁶⁹⁴-Ala⁶⁹⁵ bond and Leu⁶⁹⁷-Val⁶⁹⁸ bond, respectively, in the bait regions sequence of α₂-M. The conformation of α₂-M was also shown to be markedly altered upon inhibition of these enzymes as examined by native polyacrylamide gel electrophoresis and electron microscopy. These results show the entrapment and concomitant inhibition of those proteinases by α₂-M.     

Phenogroups and quantitative allelic expression for rabbit serum α2-macroglobulin allotypes

The four allotypic specificities, z1, z2, y3, and y4, of rabbit ₂ -macroglobulin ( ₂ M) were found to be inherited as three phenogroups, z1y3, z1y4, or z2y3. The phenogroup inheritance can be viewed as reflecting the close linkage of two loci, i.e., the Mtz locus with the z ¹ and z ² alleles and the Mty locus with the y ³ and y ⁴ alleles. Radioimmunoassay of ₂ M by an indirect precipitation method confirmed our earlier studies by a direct method showing that nearly all molecules of z1 or z2 homozygotes possess either z1 or z2, respectively, and that most molecules in the heterozygote are hybrids with both z2 and z2. Unlabeled ₂ M from homozygotes and heterozygotes was used to competitively inhibit the reaction of anti-z1 or anti-z2 with labeled ₂ M. This revealed that z1z2 heterozygotes possess approximately 35% of the z1 and 64% of the z2 determinants found in the respective homozygotes. These results resemble previous observations with the y3 and y4 allotypes which indicated an unequal contribution of allelic genes in the formation of rabbit ₂ M and the lack of allelic exclusion in most of the molecules. Unlike immunoglobulins and low-density lipoproteins, hybrid ₂ M molecules form by the interaction of homologous chromosomes.This investigation was supported in part by U.S. Public Health Service Grants AI-09241 and AI-07043. Presented in part at the 55th Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, Illinois, April 12–17, 1971.In partial fulfillment of the requirements for the Doctor of Philosophy Degree in the Graduate College of the University of Illinois. Supported by a postdoctoral fellowship from the Schweppe Foundation.Recipient of U.S. Public Health Service Research Career Development Award AI-28687.     

Differential glycosylation of α-dystroglycan and proteins other than α-dystroglycan by like-glycosyltransferase

Genetic defects in like-glycosyltransferase (LARGE) cause congenital muscular dystrophy with central nervous system manifestations. The underlying molecular pathomechanism is the hypoglycosylation of α-dystroglycan (α-DG), which is evidenced by diminished immunoreactivity to IIH6C4 and VIA4-1, antibodies that recognize carbohydrate epitopes. Previous studies indicate that LARGE participates in the formation of a phosphoryl glycan branch on O-linked mannose or it modifies complex N- and mucin O-glycans. In this study, we overexpressed LARGE in neural stem cells deficient in protein O-mannosyltransferase 2 (POMT2), an enzyme required for O-mannosyl glycosylation. The results showed that overexpressing LARGE did not lead to hyperglycosylation of α-DG in POMT2 knockout (KO) cells but did generate IIH6C4 and VIA4-1 immunoreactivity and laminin-binding activity. Additionally, overexpressing LARGE in cells deficient in both POMT2 and α-DG generated laminin-binding IIH6C4 immunoreactivity. These results indicate that LARGE expression resulted in the glycosylation of proteins other than α-DG in the absence of O-mannosyl glycosylation. The IIH6C4 immunoreactivity generated in double-KO cells was largely removed by treatment either with peptide N-glycosidase F or with cold aqueous hydrofluoric acid, suggesting that LARGE expression caused phosphoryl glycosylation of N-glycans. However, the glycosylation of α-DG by LARGE is dependent on POMT2, indicating that LARGE expression only modifies O-linked mannosyl glycans of α-DG. Thus, LARGE expression mediates the phosphoryl glycosylation of not only O-mannosyl glycans including those on α-DG but also N-glycans on proteins other than α-DG.     

Multifaceted Mechanisms of HIV-1 Entry Inhibition by Human α-Defensin

The human neutrophil peptide 1 (HNP-1) is known to block the human immunodeficiency virus type 1 (HIV-1) infection, but the mechanism of inhibition is poorly understood. We examined the effect of HNP-1 on HIV-1 entry and fusion and found that, surprisingly, this α-defensin inhibited multiple steps of virus entry, including: (i) Env binding to CD4 and coreceptors; (ii) refolding of Env into the final 6-helix bundle structure; and (iii) productive HIV-1 uptake but not internalization of endocytic markers. Despite its lectin-like properties, HNP-1 could bind to Env, CD4, and other host proteins in a glycan- and serum-independent manner, whereas the fusion inhibitory activity was greatly attenuated in the presence of human or bovine serum. This demonstrates that binding of α-defensin to molecules involved in HIV-1 fusion is necessary but not sufficient for blocking the virus entry. We therefore propose that oligomeric forms of defensin, which may be disrupted by serum, contribute to the anti-HIV-1 activity perhaps through cross-linking virus and/or host glycoproteins. This notion is supported by the ability of HNP-1 to reduce the mobile fraction of CD4 and coreceptors in the plasma membrane and to precipitate a core subdomain of Env in solution. The ability of HNP-1 to block HIV-1 uptake without interfering with constitutive endocytosis suggests a novel mechanism for broad activity against this and other viruses that enter cells through endocytic pathways.     

Functional modifications of α2-macroglobulin by primary amines. Kinetics of inactivation of α2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin

The unique steric inhibition of endopeptidases by human alpha(2)M (alpha(2)-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by alpha(2)M was closely correlated with the loss of the methylamine-reactive sites in alpha(2)M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/alpha(2)M ratios, no unaccounted loss of trypsin-binding capacity occurred. As alpha(2)M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form alpha(2)M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [(14)C]methylamine with alpha(2)M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of alpha(2)M with time of methylamine treatment. It was found that conformational change of alpha(2)M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of (125)I-labelled trypsin to alpha(2)M did follow the same kinetics as the conformational change. This discrepancy between total binding ((125)I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound (125)I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of alpha(2)M and to the mechanism of formation of the complex.     

The interaction of α2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases

1. Experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by alpha(2)-macroglobulin, as are endopeptidases of other classes. 2. A specific collagenase from rabbit synovial cells was inhibited by human serum. The inhibition could be attributed entirely to alpha(2)-macroglobulin; alpha(1)-trypsin inhibitor was not inhibitory. alpha(2)-Macroglobulin presaturated with trypsin or cathepsin B1 did not inhibit collagenase, and pretreatment of alpha(2)-macroglobulin with collagenase prevented subsequent reaction with trypsin. The binding of collagenase by alpha(2)-macroglobulin was not reversible in gel chromatography. 3. The collagenolytic activity of several rheumatoid synovial fluids was completely inhibited by incubation of the fluids with alpha(2)-macroglobulin. 4. The collagenase of human polymorphonuclear-leucocyte granules showed time-dependent inhibition by alpha(2)-macroglobulin. 5. The collagenolytic metal proteinase of Crotalus atrox venom was inhibited by alpha(2)-macroglobulin. 6. The collagenase of Clostridium histolyticum was bound by alpha(2)-macroglobulin, and inhibited more strongly with respect to collagen than with respect to a peptide substrate. 7. Thermolysin, the metal proteinase of Bacillus thermoproteolyticus, was bound and inhibited by alpha(2)-macroglobulin. 8. It was shown by polyacrylamidegel electrophoresis of reduced alpha(2)-macroglobulin in the presence of sodium dodecyl sulphate that synovial-cell collagenase, clostridial collagenase and thermolysin cleave the quarter subunit of alpha(2)-macroglobulin near its mid-point, as do serine proteinases. 9. The results are discussed in relation to previous work, and it is concluded that the characteristics of interaction of the metal proteinases with alpha(2)-macroglobulin are the same as those of other proteinases.     

Inhibition of α-synuclein aggregation by small heat shock proteins

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.     

Kinetics of the inhibition of free and elastin-bound human pancreatic elastase by α1-proteinase inhibitor and α2-macroglobulin

At pH 8.0 and 25°C α₁-proteinase inhibitor and α₂-macroglobulin bind human pancreatic elastase with rate constants of 4.7·10⁵ M⁻¹·s⁻¹ and 6.4·10⁶ M⁻¹·s⁻¹, respectively. The corresponding delay times of elastase inhibition in plasma are 0.4 s and 0.2 s, respectively, indicating that both inhibitors may act as physiological antielastases. Elastin impairs the elastase inhibitory capacity of α₁-proteinase inhibitor and α₂-macroglobulin. In presence of human elastin, the former behaves like a slow-binding elastase inhibitor, with a rate constant of about 260 M⁻¹·s⁻¹. In contrast, α₂-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin.     

Chaperone-like activity of the acute-phase component human serum α1-acid glycoprotein: Inhibition of thermal- and chemical-induced aggregation of various proteins

In vitro chaperone-like activity of the acute-phase component and plasma drug transporter human α₁-acid glycoprotein (AAG) has been shown for the first time. AAG suppressed thermal aggregation of a variety of unrelated enzymatic (e.g., aldolase, catalase, enolase, carbonic anhydrase) and non-enzymatic proteins (β-lactoglobulin, ovotransferrin) and it also prevented dithiothreitol induced aggregation of insulin. The anti-aggregation ability of AAG was abolished/reduced upon drug binding suggesting that protein–protein interactions established between the lipocalin β-barrel fold of AAG and hydrophobic surfaces of the stressed proteins are involved in the chaperone-like activity. The results shed some light on the possible biological function of this enigmatic protein and suggest that besides haptoglobin, clusterin, fibrinogen and α₂-macroglobulin AAG can be considered as a novel member of the extracellular molecular chaperones found in human body fluids.     

The interaction between α2-macroglobulin and cationic aspartate aminotransferase

On starch-gel or polyacrylamide-gel electrophoresis of human serum, a supernumerary zone of aspartate aminotransferase activity may be demonstrated, migrating with the slow alpha(2) protein zone. This appearance is due only to cationic aspartate aminotransferase, bound by alpha(2)-macroglobulin. The binding is strongly potentiated by dilute borate buffers.     

Thiol reduction of human α2-macroglobulin. The subunit structure

1. Human alpha(2)-macroglobulin was prepared from a fraction obtained during the large-scale separation of normal human plasma proteins for clinical use. 2. Sedimentation-equilibrium measurements indicated a molecular weight of 725000. A value of 18.1S was obtained for s(0) (20,w). 3. The dissociation that occurs in the pH range 4.5-2.5 and in the region of neutrality in urea-containing solutions is consistent with a dimeric structure of the molecule. 4. The effects of the thiol reagents mercaptoethanol, mercaptoethylamine and N-acetylcysteine were investigated over a range of experimental conditions. Distinct components having sedimentation coefficients of 15, 12 and 8.5S were identified. 5. Conditions were found under which limited reduction with thiol liberated a subunit with a molecular weight approximately one-quarter of that of the intact molecule. This subunit retains the serological specificity of the whole molecule.     

The isolation and partial characterization of α2-macroglobulin from the serum of the ostrich (Struthio camelus)

• 1.1. α₂-Macroglobulin (α₂M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α₂M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α₂M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
• 2.2. α₂M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn²⁺-affinity chromatography.
• 3.3. Ostrich α₂M migrated as a single band (M_r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α₂ M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
• 4.4. Isoelectric focusing revealed a pI of 5.3.
• 5.5. The amino acid composition of ostrich α₂M is typical of an α₂M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
• 6.6. α₂M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α₂M.
• 7.7. Ostrich α₂M seems to show many physical, chemical and kinetic properties similar to those of other known α₂Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.

     

Lack of association between α2-macroglobulin polymorphisms and Alzheimer's disease

This study was undertaken to investigate the role of two alpha2-macroglobulin (A2M) polymorphisms, an intronic 5-bp deletion and Ile1000Val, in the development of Alzheimer's disease (AD) and to evaluate the interaction between the apolipoprotein E (APOE) and A2M polymorphisms. The A2M polymorphisms were screened by using polymerase-chain-reaction-based assays in 555 white late-onset AD cases and 446 controls. The gentoype distributions of the 5-bp deletion and Ile1000Val polymorphisms were comparable between cases and controls (P = 0.158 and P = 0.148, respectively). Likewise, there was no significant difference in allele frequencies of each polymorphism among cases and controls (P = 0.361 and P = 0.062, respectively). The stratification of data by APOE*4 status also did not yield any significant association. In conclusion, we observed no association between either the intronic deletion polymorphism or the Ile1000Val polymorphism of A2M and AD in our case-control cohort.     

Nitrite Reductase Activity and Inhibition of H2S Biogenesis by Human Cystathionine ?-Synthase

Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS) as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe^II-NO) CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR) and nitrite. Formation of Fe^II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.     

Inhibition of protein tyrosine phosphatase (PTP1B) and α-glucosidase by geranylated flavonoids from Paulownia tomentosa

Protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase are important targets to treat obesity and diabetes, due to their deep correlation with insulin and leptin signalling, and glucose regulation. The methanol extract of Paulownia tomentosa fruits showed potent inhibition against both enzymes. Purification of this extract led to eight geranylated flavonoids (1–8) displaying dual inhibition of PTP1B and α-glucosidase. The isolated compounds were identified as flavanones (1–5) and dihydroflavonols (6–8). Inhibitory potencies of these compounds varied accordingly, but most of the compounds were highly effective against PTP1B (IC₅₀?=?1.9–8.2?μM) than α-glucosidase (IC₅₀?=?2.2–78.9?μM). Mimulone (1) was the most effective against PTP1B with IC₅₀?=?1.9?μM, whereas 6-geranyl-3,3′,5,5′,7-pentahydroxy-4′-methoxyflavane (8) displayed potent inhibition against α-glucosidase (IC₅₀?=?2.2?μM). All inhibitors showed mixed type Ι inhibition toward PTP1B, and were noncompetitive inhibitors of α-glucosidase. This mixed type behavior against PTP1B was fully demonstrated by showing a decrease in V_max, an increase of K_m, and K_ik/K_iv ratio ranging between 2.66 and 3.69.     

Inhibition and disaggregation of α-synuclein oligomers by natural polyphenolic compounds

Aggregation of alpha-synuclein (αS) into oligomers is critically involved in the pathogenesis of Parkinson's disease (PD). Using confocal single-molecule fluorescence spectroscopy, we have studied the effects of 14 naturally-occurring polyphenolic compounds and black tea extract on αS oligomer formation. We found that a selected group of polyphenols exhibited potent dose-dependent inhibitory activity on αS aggregation. Moreover, they were also capable of robustly disaggregating pre-formed αS oligomers. Based upon structure-activity analysis, we propose that the key molecular scaffold most effective in inhibiting and destabilizing self-assembly by αS requires: (i) aromatic elements for binding to the αS monomer/oligomer and (ii) vicinal hydroxyl groups present on a single phenyl ring. These findings may guide the design of novel therapeutic drugs in PD.     

Evolution of α2-macroglobulin. The structure of a protein homologous with human α2-macroglobulin from plaice (Pleuronectes platessa L.) plasma

The plaice (Pleuronectes platessa L.) papain-binding protein previously demonstrated to be homologous with human alpha(2)-macroglobulin, and designated plaice alpha(2)-macroglobulin homologue or alphaMh, was shown to be a glycoprotein of s(20,w) 11.86S. In polyacrylamide-gel pore-limit electrophoresis under non-denaturing conditions plaice alphaMh migrated to the same position as half-molecules of human alpha(2)-macroglobulin, and treatment with methylamine or a proteinase caused no change in its electrophoretic properties. Either denaturation in urea (4m) or mild reduction by dithiothreitol (1mm) partially dissociated plaice alphaMh into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions plaice alphaMh dissociated into subunits of M(r) 105000 (I) and 90000 (II). Approximately equal amounts of each subunit were formed, and peptide ;mapping' showed subunits I and II to be distinct polypeptide chains. Under alkaline denaturing conditions, a proportion of the I chains of alphaMh were cleaved into fragments of M(r) about 60000 and 40000. This cleavage was favoured by reducing conditions and prevented by prior inactivation of the alphaMh with methylamine. [(14)C]Methylamine allowed to react with alphaMh became covalently linked to subunit I. These properties suggested the existence of an autolytic site on subunit I analogous to the autolytic site of human alpha(2)-macroglobulin. Reaction of alphaMh with a proteinase resulted in cleavage of a fragment of M(r) 10000-15000 from subunit I. A proportion of the proteinase molecules trapped by alphaMh became covalently linked to the inhibitor. A scheme is proposed for the evolution of human alpha(2)-macroglobulin and plaice alphaMh from a common ancestral protein, which may also have been an ancestor of complement components C3 and C4.