Cloning, expression, purification, and characterization of rat MMP-12

Macrophage metalloelastase (MMP-12) is implicated in the pathology of many diseases such as emphysema, aortic lesions and cancer. Recently, MMP-12 was cloned and purified from mouse and human macrophages. We report here the expression of the full-length and catalytic domain of rat MMP-12 in Escherichia coli and characterization of the purified enzyme. Inclusion bodies of expressed rat MMP-12 catalytic domain were denatured and refolded using a new method, and then affinity purified to near homogeneity with zinc-chelating Sepharose. The purified rat MMP-12 catalytic domain was highly active in digesting substrates, having a K(m) of 12 microM and optimal pH of 7.5--8.5. During investigation of natural substrate specificity, we found that rat MMP-12 catalytic domain was able to completely degrade collagen-V, partially degrade collagen-I, but it was unable to digest collagen-IV. The enzyme could also degrade osteonectin, vitronectin, and fibronectin, but not laminin and albumin. The catalytic properties and natural substrate specificity of rat MMP-12 catalytic domain differed from those of human MMP-12 catalytic domain.     

Crystal structure of human macrophage elastase (MMP-12) in complex with a hydroxamic acid inhibitor

Human macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) that plays, like other members of the family, an important role in inflammatory processes contributing to tissue remodelling and destruction. In particular, a prominent role of MMP-12 in the destruction of elastin in the lung alveolar wall and the pathogenesis of emphysema has been suggested. It is therefore an attractive therapeutic target. We describe here the crystal structure of the catalytic domain of MMP-12 in complex with a hydroxamic acid inhibitor, CGS27023A. MMP-12 adopts the typical MMP fold and binds a structural zinc ion and three calcium ions in addition to the catalytic zinc ion. The enzyme structure shows an ordered N terminus close to the active site that is identical in conformation with the superactivated form of MMP-8. The S1'-specificity pocket is large and extends into a channel through the protein, which puts MMP-12 into the class of MMPs 3, 8 and 13 with large and open specificity pockets. The two crystallographically independent molecules adopt different conformations of the S1'-loop and its neighbouring loop due to differing crystal packing environments, suggesting that flexibility or the possibility of structural adjustments of these loop segments are intrinsic features of the MMP-12 structure and probably a common feature for all MMPs. The inhibitor binds in a bidentate fashion to the catalytic zinc ion. Its polar groups form hydrogen bonds in a substrate-like manner with beta-strand sIV of the enzyme, while the hydrophobic substituents are either positioned on the protein surface and are solvent-exposed or fill the upper part of the specificity pocket. The present structure enables us to aid the design of potent and selective inhibitors for MMP-12.     

Protein disulfide isomerase-mediated disulfide bonds regulate the gelatinolytic activity and secretion of matrix metalloproteinase-9

Matrix metalloproteinase-9 (MMP-9) is one of the major MMPs that can degrade extracellular matrix. Besides normal physiological functions, MMP-9 is involved in metastasis and tumor angiogenesis. Although several inhibitors of MMP-9 have been identified, in vivo regulators of MMP-9 activation are unknown. In the present study we intended to investigate novel therapeutic target protein(s) that regulate MMP-9 activation and/or secretion. We have identified protein disulfide isomerase as a novel upstream regulator of MMP-9. Mass spectrometric analysis of post-translational modification in MMP-9 confirmed six disulfide bonds in the catalytic domain and one disulfide bond in the hemopexin domain of MMP-9. Establishment of cells that overexpressed wild-type and mutant forms of MMP-9 revealed that 'cysteine-switch' and disulfide bonds within the catalytic domain are necessary for the secretion and intracellular trafficking of MMP-9. However, the disulfide bond of the hemopexin domain and other cysteines have no significant role in secretion. These insights into the secretion of MMP-9 constitute the basis for the development of potential drugs against metastasis.     

Hydrogen exchange monitored by MALDI-TOF mass spectrometry for rapid characterization of the stability and conformation of proteins

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to monitor hydrogen exchange on entire proteins. Two alternative methods have been used to carry out the hydrogen exchange studies, exchanging deuteron (H to D experiments) or proton (D to H experiments). In the former case, the use of a deuterated matrix has made possible to overcome back-exchange problems and attain reproducible results. The methods presented have been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to differentially compare the activation domain of human procarboxypeptidase A2 versus three site-directed mutants of different conformational stability. In this work, we show that by using MALDI-TOF MS to monitor hydrogen exchange in entire proteins, it is possible to rapidly check the folding state of a protein and characterize mutational effects on protein conformation and stability, while requiring minimal amounts of sample.     

Matrix Metalloproteinase-3 Is Increased and Participates in Neuronal Apoptotic Signaling Downstream of Caspase-12 during Endoplasmic Reticulum Stress

Although endoplasmic reticulum (ER) stress-induced apoptosis has been associated with pathogenesis of neurodegenerative diseases, the cellular components involved have not been well delineated. The present study shows that matrix metalloproteinase (MMP)-3 plays a role in the ER stress-induced apoptosis. ER stress induced by brefeldin A (BFA) or tunicamycin (TM) increases gene expression of MMP-3, selectively among various MMP subtypes, and the active form of MMP-3 (actMMP-3) in the brain-derived CATH.a cells. Pharmacological inhibition of enzyme activity, small interference RNA-mediated gene knockdown, and gene knock-out of MMP-3 all provide protection against ER stress. MMP-3 acts downstream of caspase-12, because both pharmacological inhibition and gene knockdown of caspase-12 attenuate the actMMP-3 increase, but inhibition and knock-out of MMP-3 do not alter caspase-12. Furthermore, independently of the increase in the protein level, the catalytic activity of MMP-3 enzyme can be increased via lowering of its endogenous inhibitor protein TIMP-1. Caspase-12 causes liberation of MMP-3 enzyme activity by degrading TIMP-1 that is already bound to actMMP-3. TIMP-1 is decreased in response to ER stress, and TIMP-1 overexpression leads to cell protection and a decrease in MMP-3 activity. Taken together, actMMP-3 protein level and catalytic activity are increased following caspase-12 activation during ER stress, and this in turn plays a role in the downstream apoptotic signaling in neuronal cells. MMP-3 and TIMP-1 may therefore serve as cellular targets for therapy against neurodegenerative diseases.     

CUEDC2中CUE结构域的表达纯化及结构初探

目的:建立CUEDC2中CUE结构域的原核表达系统,获得13C、15N同位素标记的CUE结构域蛋白质,以用于结构生物学研究。方法:利用分子生物学方法将CUE结构域编码序列构建到pET-28a原核表达系统,表达和纯化13C、15N标记的重组蛋白;用SDS-PAGE等方法对其进行鉴定。结果:目的蛋白经SDS-PAGE和MALDI-TOF/MS检测,相对分子质量正确,圆二色谱和核磁共振波谱结果显示目的蛋白折叠良好。结论:获得了高浓度、高纯度、折叠良好的CUE结构域标记蛋白质,利于进一步的结构生物学研究。     

Mass spectrometric characterization of proteins from the SARS virus: a preliminary report

A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a approximately 46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted approximately 139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS.     

Hydroxamate-based peptide inhibitors of matrix metalloprotease 2

There is major interest in designing inhibitors for matrix metalloproteinase 2 (MMP-2, gelatinase A) since this enzyme is known to be involved in pathological processes such as tumor invasion or rheumatoid arthritis. The majority of MMP-2 inhibitor candidate drugs block the active site of MMP-2 by binding to its catalytic Zn2+ ion through a chelating (hydroxamate, sulphonate etc.) group. Despite the general interest in designing MMP-2 inhibitors, the results with many of the drug candidates were disappointing, their failure was usually explained by cross-reactions with other MMPs. One way to enhance MMP-2 selectivity is to design inhibitors that interact with both the active site and exosites such as the fibronectin type II (FN2) domains of the enzyme. In the present work, we have examined the inhibitory potential and MMP-2 selectivity of hydroxamates of three groups of peptides known to bind to the collagen-binding FN2 domains of MMP-2. The first type of peptides consisted of collagen-like (Pro-Pro-Gly)(n) repeats, peptides of the second group were identified from a random 15-mer phage display library based on their binding to immobilized FN2 domains of MMP-2. A hydroxamate of peptide p33-42, known to bind to the third FN2 domain of MMP-2 has also been tested. Our studies have shown that these compounds inhibited MMP-2 with IC50 values of 10-100 microM. The fact that their inhibitory potential was nearly identical for MMP-2del, a recombinant version of MMP-2 that lacks the FN2 domains, suggests that inhibition is not mediated by their binding to FN2 domains. It seems likely that the failure to exploit interaction with the FN2 domains is due to the fact that the FN2 domains and the catalytic domain of MMP-2 tumble independently, therefore only a tiny fraction of the conformational isomers can bind peptide hydroxamates via both the active site and the FN2 domain(s).     

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.     

MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III

Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly₇₇₅-Leu₇₇₆- in α-2 type I collagen and -Gly₇₇₅-Ile₇₇₆- in α-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III.     

Molecular characterization of Alg8, a putative glycosyltransferase, involved in alginate polymerisation

The topology of Alg8, the proposed catalytic subunit of the alginate polymerase, was assessed using PhoA and LacZ fusion protein analysis. This analysis suggested that the periplasmic loop comprises only three amino acid residues with the adjacent transmembrane helices at positions 361–387 and 393–416. Accordingly, the extended cytosolic loop could be located at positions 71–361 and was proposed to contain important catalytic residues. Further experimental evidence for this cytosolic domain was obtained by independently demonstrating this protein region as purified soluble protein domain. The soluble protein domain was identified by MALDI-TOF/MS and presumably represents the cytosolic catalytic domain of Alg8. Site-directed mutagenesis of 11 conserved residues in the cytosolic loop showed that D-188/D-190 (DXD motif), D-295/D-296 (acid–base catalysts) and K-297 were each essential for in vivo polymerase activity, whereas D-179/D-181 (DXD motif), C-244, R-263, D-279, and E-282 were not directly involved in the polymerisation reaction. The role of these amino acid residues with respect to the catalysed alginate polymerisation reaction was discussed with the aid of the recently developed structural model of Alg8.     

Matrix metalloproteinase interactions with collagen and elastin

Most abundant in the extracellular matrix are collagens, joined by elastin that confers elastic recoil to the lung, aorta, and skin. These fibrils are highly resistant to proteolysis but can succumb to a minority of the matrix metalloproteinases (MMPs). Considerable inroads to understanding how such MMPs move to the susceptible sites in collagen and then unwind the triple helix of collagen monomers have been gained. The essential role in unwinding of the hemopexin-like domain of interstitial collagenases or the collagen binding domain of gelatinases is highlighted. Elastolysis is also facilitated by the collagen binding domain in the cases of MMP-2 and MMP-9, and remote exosites of the catalytic domain in the case of MMP-12.     

Identification of Specific Hemopexin-like Domain Residues That Facilitate Matrix Metalloproteinase Collagenolytic Activity

Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Several members of the matrix metalloproteinase (MMP) family catalyze the hydrolysis of collagen triple helical structure. This study has utilized triple helical peptide (THP) substrates and inhibitors to dissect MMP-1 collagenolytic behavior. Analysis of MMP-1/THP interactions by hydrogen/deuterium exchange mass spectrometry followed by evaluation of wild type and mutant MMP-1 kinetics led to the identification of three noncatalytic regions in MMP-1 (residues 285–295, 302–316, and 437–457) and two specific residues (Ile-290 and Arg-291) that participate in collagenolysis. Ile-290 and Arg-291 contribute to recognition of triple helical structure and facilitate both the binding and catalysis of the triple helix. Evidence from this study and prior studies indicates that the MMP-1 catalytic and hemopexin-like domains collaborate in collagen catabolism by properly aligning the triple helix and coupling conformational states to facilitate hydrolysis. This study is the first to document the roles of specific residues within the MMP-1 hemopexin-like domain in substrate binding and turnover. Noncatalytic sites, such as those identified here, can ultimately be utilized to create THP inhibitors that target MMPs implicated in disease progression while sparing proteases with host-beneficial functions.The mechanism of collagenolysis, by which proteases catalyze the hydrolysis of amide bonds within triple helical structures, has been investigated for over 30 years. Despite this lengthy period, few inroads have been made in the identification of specific enzyme residues that facilitate collagenolysis. The primary mammalian collagenases have been identified as cathepsin K and several members of the matrix metalloproteinase (MMP)³ family. Most of the early work on MMP collagenolysis focused on analysis of the sites of hydrolysis, and how unique features within these sites may direct collagen catabolism (1). More recent work has evaluated the active sites and domains of MMPs to better understand the dynamic role that the enzyme plays in collagen hydrolysis (2–4).Collagenolytic members of the MMP family possess similar domain organizations, including propeptide, catalytic (CAT), linker, and hemopexin-like (HPX) domains (5). Several of these domains and/or regions within them have been implicated in collagenolysis. For example, MMP-1 residues 183–191, which are on the V-B loop between the fifth β-strand and the second α-helix in the CAT domain, as well as the active site cleft itself, have substantial roles in collagenolysis (6, 7). MMP-1 residue Gly-233 has been implicated as necessary for conformational flexibility of the active site (8). Within the MMP-1 linker domain, residues 262–276 were proposed to form a polyproline type II helix and interact with and destabilize the MMP cleavage site in collagen (9), whereas Gly-272 may allow bending of the linker domain to aid in interaction between the CAT and HPX domains (10).The HPX domain has a critical role in collagenolysis, as removal of the MMP-1, MMP-8, MMP-13, or MMP-14 (MT1-MMP) HPX domain results in a loss of collagenolytic activity (11–16). However, no information has been obtained as to the identity of specific residues within the HPX domain that participate in collagenolysis. Secondary binding sites (exosites) may promote interaction of proteases with large, macromolecular substrates, such as collagen. The identification of exosites involved in collagenolysis may aid in the design of selective MMP inhibitors (17–20). Ultimately, as exosites are identified, the manner in which the CAT, linker, and HPX domains work together to facilitate collagenolysis can be revealed.One approach for the rapid analysis of protein structure and identification of binding sites within proteins involves hydrogen/deuterium exchange (HDX) of protein backbone amide hydrogens with detection by mass spectrometry (MS) (21–23). A protein or protein/ligand pair is incubated for defined intervals in a deuterated environment. After rapid quenching of the HDX reaction, the partially deuterated protein is digested, and the resulting peptide fragments are analyzed by LC-MS. The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. HDX MS has been used previously to monitor the interaction between doxycycline and MMP-7 (24). The interaction sites identified were consistent with other biophysical studies mapping doxycycline binding outside of the catalytic Zn²⁺ (24). This present study has utilized HDX MS with a triple helical peptide (THP) substrate to identify nonactive site MMP-1 regions involved in collagenolysis. Subsequently, site-specific mutagenesis of MMP-1 in combination with THP inhibitors and substrates was utilized to identify, for the first time, specific HPX domain residues that participate in collagenolysis and to provide insight as to how these residues function mechanistically.     

Affinity-tagged phosphorylation assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ATPA-MALDI): application to calcium/calmodulin-dependent protein kinase

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.     

Apolactoferrin inhibits the catalytic domain of matrix metalloproteinase-2 by zinc chelation.

Lactoferrin (LTF) is a multifunctional iron-binding protein that is also capable of binding other divalent metal cations, especially Zn2+. Recent investigations indicate that lactoferrin levels are elevated in many disease conditions in which matrix metalloproteinases (MMPs), particularly MMP-2, are also elevated, suggesting that the 2 proteins may interact. This possibility was examined by determining the effect of LTF in its holo (metal-bound) and apo (metal-free) forms on the proteolytic activity of MMP-2 and other similar zinc metalloproteases. Pre-incubation with apolactoferrin, but not hololactoferrin, greatly reduced the hydrolysis of a peptide substrate by MMP-2, but not by MMP-1, -8, -9, or -13. This inhibition was specific for the 42 kDa catalytic domain fragment of MMP-2 lacking the hemopexin domain, since the 66 kDa form was poorly inhibited by apolactoferrin. The inhibition of the MMP-2 catalytic domain was strongly temperature sensitive, indicating that the conformation of one or both proteins is crucial to this interaction. To ascertain the mechanism of inhibition, increasing concentrations of ZnCl2 and FeCl2 were added to the reaction. While addition of Fe2+ did not reverse inhibition, the addition of Zn2+ resulted in a recovery of MMP-2 activity, and furthermore, zinc-saturated LTF did not inhibit MMP-2. Together, these data strongly suggest that apolactoferrin is capable of removing the catalytic zinc from the active site of MMP-2, although an exosite-based interaction between the 2 proteins cannot be fully ruled out. This inhibitory activity suggests a novel function for LTF and may represent a novel regulatory mechanism that regulates proteolysis by MMP-2 in vivo.     

Mapping of macrophage elastase cleavage sites in insoluble human skin elastin

Macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) and is active against multiple extracellular protein substrates such as elastin. Its effect on elastin is central to emphysema in the lung and photoaging of skin. Its expression in the skin increases on photodamaged skin and upon aging. Detecting and characterizing peptides cleaved in elastin, therefore, helps to understand such degradative disease processes in the skin and is also needed to assist in the rational design of agents that specifically inhibit the degradation. In this study, cleavage sites of MMP-12 in human skin elastin were extensively investigated. The peptides formed as a result of cleavages by this enzyme in the human skin elastin were characterized using mass spectrometry. A total of 41 peptides ranging from 4 to 41 amino acids were identified and 36 cleavage sites were determined. Amino acids encoded by exons 5, 6, 26, 28-31 were particularly susceptible to cleavages by MMP-12 and none or very few cleavages were detected from domains encoded by the remaining exons. The amino acid preferences of the different subsites on the catalytic domain of MMP-12 were analyzed.     

Structural characterization and binding properties of the hemopexin-like domain of the matrixmetalloproteinase-19

The matrixmetalloproteinase-19 (MMP-19) belongs to the superfamily of the zinc-dependent endopeptidases, which are secreted by cells and are involved in the remodeling of the extracellular matrix. The full-length protein consists of a signal peptide, a propeptide, a catalytic domain and a C-terminal hemopexin-like domain. For other members of this superfamily, the hemopexin-like domain has been described to be involved in substrate recognition. In this study, the hemoxpexin domain of MMP-19 was expressed in Escherichia coli, refolded, and purified. For structural characterization, circular dichroism and NMR spectroscopy were used. We show that the hemopexin-like domain of MMP-19 is able to bind calcium and this binding induces a conformational change and an increase in the thermal stability of the domain. MMP-19 promotes proliferation of keratinocytes by cleaving the insulin-like-growth factor binding protein-3, thereby causing the release of IGF-1, which is a potent growth factor for these cells. By plasmon resonance experiments, we show that the isolated hemopexin-like domain is able to bind to the insulin-like-growth factor binding protein-3. These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists.     

Solution structure and backbone dynamics of the catalytic domain of matrix metalloproteinase-2 complexed with a hydroxamic acid inhibitor

MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 A from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1' specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1' specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.     

Sub-proteome analysis of novel IgE-binding proteins from Bermuda grass pollen

Bermuda grass (Cynodon dactylon) pollen (BGP) is one of the most common causes of airway allergic disease, and has been shown to contain over 12 allergenic proteins on 1-D immunoglobulin E (IgE) immunoblots. However, only a few allergens have been identified and characterized. Cyn d 1 is a major allergen and the most abundant protein in BGP, representing 15% of the whole-pollen extract. To investigate variability in the IgE-reactive patterns of BGP-sensitized patients and to identify other prevalent allergens, a BGP extract was passed through an affinity column to remove Cyn d 1, and the non-bound material was collected and analyzed by 2-DE. IgE-reactive proteins were subsequently characterized by immunoblotting using serum samples from ten BGP-allergic patients. The prevalent IgE-reactive proteins were identified by MALDI-TOF MS, N-terminal sequence similarity, and LC-MS/MS. Here, we present a sub-proteome approach for allergen investigation and its use for determining BGP 2-DE profiles and identifying six novel allergens.