Publications by authors named "Tomas Koudelka"

36 Publications

Phosphorylation of meprin β controls its cell surface abundance and subsequently diminishes ectodomain shedding.

FASEB J 2021 07;35(7):e21677

Biochemical Institute, Unit for Degradomics of the Protease Web, University of Kiel, Kiel, Germany.

Meprin β is a zinc-dependent metalloprotease exhibiting a unique cleavage specificity with strong preference for acidic amino acids at the cleavage site. Proteomic studies revealed a diverse substrate pool of meprin β including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprin β is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprin β including interactors, sheddases, and activators has been intensively investigated while intracellular regulation has been barely addressed in the literature. This study aimed to analyze C-terminal phosphorylation of meprin β with regard to cell surface expression and proteolytic activity. By immunoprecipitation of endogenous meprin β from the colon cancer cell line Colo320 and subsequent LC-MS analysis, we identified several phosphorylation sites in its C-terminal region. Here, T694 in the C-terminus of meprin β was the most preferred residue after phorbol 12-myristate 13-acetate (PMA) stimulation. We further demonstrated the role of protein kinase C (PKC) isoforms for meprin β phosphorylation and identified the involvement of PKC-α and PKC-β. As a result of phosphorylation, the meprin β activity at the cell surface is reduced and, consequently, the extent of substrate cleavage is diminished. Our data indicate that this decrease of the surface activity is caused by the internalization and degradation of meprin β.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.202100271RDOI Listing
July 2021

ADP-Ribosylation Regulates the Signaling Function of IFN-γ.

Front Immunol 2021 8;12:642545. Epub 2021 Mar 8.

Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2021.642545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7983947PMC
March 2021

Cell Surface Processing of CD109 by Meprin β Leads to the Release of Soluble Fragments and Reduced Expression on Extracellular Vesicles.

Front Cell Dev Biol 2021 2;9:622390. Epub 2021 Mar 2.

Anatomical Institute, Christian-Albrechts-University Kiel, Kiel, Germany.

Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on primitive hematopoietic stem cells, activated platelets, CD4 and CD8 T cells, and keratinocytes. In recent years, CD109 was also associated with different tumor entities and identified as a possible future diagnostic marker linked to reduced patient survival. Also, different cell signaling pathways were proposed as targets for CD109 interference including the TGFβ, JAK-STAT3, YAP/TAZ, and EGFR/AKT/mTOR pathways. Here, we identify the metalloproteinase meprin β to cleave CD109 at the cell surface and thereby induce the release of cleavage fragments of different size. Major cleavage was identified within the bait region of CD109 residing in the middle of the protein. To identify the structural localization of the bait region, homology modeling and single-particle analysis were applied, resulting in a molecular model of membrane-associated CD109, which allows for the localization of the newly identified cleavage sites for meprin β and the previously published cleavage sites for the metalloproteinase bone morphogenetic protein-1 (BMP-1). Full-length CD109 localized on extracellular vesicles (EVs) was also identified as a release mechanism, and we can show that proteolytic cleavage of CD109 at the cell surface reduces the amount of CD109 sorted to EVs. In summary, we identified meprin β as the first membrane-bound protease to cleave CD109 within the bait region, provide a first structural model for CD109, and show that cell surface proteolysis correlates negatively with CD109 released on EVs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2021.622390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960916PMC
March 2021

Native disulphide-linked dimers facilitate amyloid fibril formation by bovine milk α-casein.

Biophys Chem 2021 03 15;270:106530. Epub 2020 Dec 15.

Research School of Chemistry, The Australian National University, Acton, ACT 2601, Australia. Electronic address:

Bovine milk α-casein, an intrinsically disordered protein, readily forms amyloid fibrils in vitro and is implicated in the formation of amyloid fibril deposits in mammary tissue. Its two cysteine residues participate in the formation of either intra- or intermolecular disulphide bonds, generating monomer and dimer species. X-ray solution scattering measurements indicated that both forms of the protein adopt large, spherical oligomers at 20 °C. Upon incubation at 37 °C, the disulphide-linked dimer showed a significantly greater propensity to form amyloid fibrils than its monomeric counterpart. Thioflavin T fluorescence, circular dichroism and infrared spectra were consistent with one or both of the dimer isomers (in a parallel or antiparallel arrangement) being predisposed toward an ordered, amyloid-like structure. Limited proteolysis experiments indicated that the region from Ala to Lys is incorporated into the fibril core, implying that this region, which is predicted by several algorithms to be amyloidogenic, initiates fibril formation of α-casein. The partial conservation of the cysteine motif and the frequent occurrence of disulphide-linked dimers in mammalian milks despite the associated risk of mammary amyloidosis, suggest that the dimeric conformation of α-casein is a functional, yet amyloidogenic, structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpc.2020.106530DOI Listing
March 2021

N-Terminomics for the Identification of In Vitro Substrates and Cleavage Site Specificity of the SARS-CoV-2 Main Protease.

Proteomics 2021 01 17;21(2):e2000246. Epub 2020 Nov 17.

Systematic Proteome Research and Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, 24105, Germany.

The genome of coronaviruses, including SARS-CoV-2, encodes for two proteases, a papain like (PL ) protease and the so-called main protease (M ), a chymotrypsin-like cysteine protease, also named 3CL or non-structural protein 5 (nsp5). M is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that M proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS-CoV, SARS-CoV-2, and hCoV-NL63 coronviruses, an LC-MS based N-terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS-CoV-2 M , 445 cleavage events from more than 300 proteins are identified, while 151 and 331 M derived cleavage events are identified for SARS-CoV and hCoV-NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.202000246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645863PMC
January 2021

Meprin β cleaves TREM2 and controls its phagocytic activity on macrophages.

FASEB J 2020 05 1;34(5):6675-6687. Epub 2020 Apr 1.

Unit for Degradomics of the Protease Web, Biochemical Institute, University of Kiel, Kiel, Germany.

The triggering receptor expressed on myeloid cells 2 (TREM2) is a multifunctional surface protein that affects survival, migration, and phagocytic capacity of myeloid cells. Soluble TREM2 levels were found to be increased in early stages of sporadic and familial Alzheimer's disease (AD) probably reflecting a defensive microglial response to some initial brain damage. The disintegrin and metalloproteases (ADAM) 10 and 17 were identified as TREM2 sheddases. We demonstrate that meprin β is a direct TREM2 cleaving enzyme using ADAM10/17 deficient HEK293 cells. LC-MS/MS analysis of recombinant TREM2 incubated with meprin β revealed predominant cleavage between Arg136 and Asp137, distant to the site identified for ADAM10/17. We further demonstrate that the metalloprotease meprin β cleaves TREM2 on macrophages concomitant with decreased levels of soluble TREM2 in the serum of Mep1b mice compared to WT controls. Isolated BMDMs from Mep1b mice showed significantly increased full-length TREM2 levels and enhanced phagocytosis efficiency compared to WT cells. The diminished constitutive shedding of TREM2 on meprin β deficient macrophages could be rescued by ADAM stimulation through LPS treatment. Our data provide evidence that meprin β is a TREM2 sheddase on macrophages and suggest that multiple proteases may be involved in the generation of soluble TREM2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201902183RDOI Listing
May 2020

Distance dependent shedding of IL-6R.

Biochem Biophys Res Commun 2020 05 26;526(2):355-360. Epub 2020 Mar 26.

Department of Structural Biology, Institute of Zoology, Am Botanischen Garten 1-9, 24118, Kiel, Germany.

Proteolytic processing of membrane proteins by A disintegrin and metalloprotease-17 (ADAM17) is a key regulatory step in many physiological and pathophysiological processes. This so-called shedding is essential for development, regeneration and immune defense. An uncontrolled ADAM17 activity promotes cancer development, chronic inflammation and autoimmune diseases. Consequently, the ADAM17 activity is tightly regulated. As a final trigger for the shedding event a phosphatidylserine (PS) flip to the outer leaflet of the cell membrane was recently described. PS interacts with the extracellular part of ADAM17, which results in the shedding event by shifting the catalytic domain towards the membrane close to the cleavage sites within ADAM17 substrates. Our data indicate that the intrinsic proteolytic activity of the catalytic domain is prerequisite for the shedding activity and constantly present. However, the accessibility for substrate cleavage sites is controlled on several levels. In this report, we demonstrate that the positioning of the catalytic domain towards the cleavage sites is a crucial part of the shedding process. This finding contributes to the understanding of the complex and multilayered regulation of ADAM17 at the cell surface.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2020.03.093DOI Listing
May 2020

Proteomic investigation of the blue mussel larval shell organic matrix.

J Struct Biol 2019 12 7;208(3):107385. Epub 2019 Sep 7.

GEOMAR Helmholtz Centre for Ocean Research, 24105 Kiel, Germany; Sven Lovén Centre for Marine Infrastructure, Gothenburg University, 45178 Fiskebäckskil, Sweden.

Shell matrix proteins (SMPs) are occluded within molluscan shells and are fundamental to the biological control over mineralization. While many studies have been performed on adult SMPs, those of larval stages remain largely undescribed. Therefore, this study aimed to characterize the larval shell proteome of the blue mussel for the first time and to compare it to adult mussel shell proteomes. Following development of a method for cleaning larval shells of tissue contaminants, 49 SMPs were identified using shotgun proteomics. Twenty-one proteins were independently identified in all samples indicating that they form a subset of the core larval shell proteome. These included: the blue mussel shell protein, a peroxidase domain-containing sequence, a laminin G domain-containing sequence, a ZIP domain-containing sequence and a ferric-chelate reductase 1-like sequence. Additional SMP domains identified were: fibronectin type III, BPTI/Kunitz, chitin-binding type 3, thyroglobulin and EF-hand. While key predictable molluscan shell matrix functions are identified, 67% of sequences remain unknown or uncharacterized, indicating that this shell proteome is unique to mussel larvae. Specifically, comparison with adult mytilids reveals that nine domains are exclusive to the larval shell proteome and only four domains are conserved among species and developmental stages. Thus, strong species-specific and ontogenetic variation exists in shell proteome composition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsb.2019.09.002DOI Listing
December 2019

Meprin β induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

FASEB J 2019 11 9;33(11):11925-11940. Epub 2019 Aug 9.

Institute of Biochemistry, University of Kiel, Kiel, Germany.

Meprin β is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin β, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin β substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin β and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin β in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin β caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin β and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin β and additional measurement of TNF-α shedding on bone marrow-derived macrophages, meprin β/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin β with ADAM proteases to control protease activities at the cell surface as part of the protease web.-Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin β induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201801371RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902663PMC
November 2019

Diazirine-functionalized mannosides for photoaffinity labeling: trouble with FimH.

Beilstein J Org Chem 2018 24;14:1890-1900. Epub 2018 Jul 24.

Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, Otto-Hahn-Platz 3/4, 24118 Kiel, Germany.

Photoaffinity labeling is frequently employed for the investigation of ligand-receptor interactions in solution. We have employed an interdisciplinary methodology to achieve facile photolabeling of the lectin FimH, which is a bacterial protein, crucial for adhesion, colonization and infection. Following our earlier work, we have here designed and synthesized diazirine-functionalized mannosides as high-affinity FimH ligands and performed an extensive study on photo-crosslinking of the best ligand (mannoside ) with a series of model peptides and FimH. Notably, we have employed high-performance mass spectrometry to be able to detect radiation results with the highest possible accuracy. We are concluding from this study that photolabeling of FimH with sugar diazirines has only very limited success and cannot be regarded a facile approach for covalent modification of FimH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3762/bjoc.14.163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071696PMC
July 2018

In vivo regulation of the A disintegrin and metalloproteinase 10 (ADAM10) by the tetraspanin 15.

Cell Mol Life Sci 2018 09 8;75(17):3251-3267. Epub 2018 Mar 8.

Institute of Biochemistry, Christian Albrechts University Kiel, Olshausenstrasse 40, 24118, Kiel, Germany.

A disintegrin and metalloproteinase 10 (ADAM10) plays a major role in the ectodomain shedding of important surface molecules with physiological and pathological relevance including the amyloid precursor protein (APP), the cellular prion protein, and different cadherins. Despite its therapeutic potential, there is still a considerable lack of knowledge how this protease is regulated. We have previously identified tetraspanin15 (Tspan15) as a member of the TspanC8 family to specifically associate with ADAM10. Cell-based overexpression experiments revealed that this binding affected the maturation process and surface expression of the protease. Our current study shows that Tspan15 is abundantly expressed in mouse brain, where it specifically interacts with endogenous ADAM10. Tspan15 knockout mice did not reveal an overt phenotype but showed a pronounced decrease of the active and mature form of ADAM10, an effect which augmented with aging. The decreased expression of active ADAM10 correlated with an age-dependent reduced shedding of neuronal (N)-cadherin and the cellular prion protein. APP α-secretase cleavage and the expression of Notch-dependent genes were not affected by the loss of Tspan15, which is consistent with the hypothesis that different TspanC8s cause ADAM10 to preferentially cleave particular substrates. Analyzing spine morphology revealed no obvious differences between Tspan15 knockout and wild-type mice. However, Tspan15 expression was elevated in brains of an Alzheimer's disease mouse model and of patients, suggesting that upregulation of Tspan15 expression reflects a cellular response in a disease state. In conclusion, our data show that Tspan15 and most likely also other members of the TspanC8 family are central modulators of ADAM10-mediated ectodomain shedding in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-018-2791-2DOI Listing
September 2018

GRP78 protects a disintegrin and metalloprotease 17 against protein-disulfide isomerase A6 catalyzed inactivation.

FEBS Lett 2017 11 11;591(21):3567-3587. Epub 2017 Oct 11.

Institute of Biochemistry, Christian-Albrechts University, Kiel, Germany.

The shedding of ectodomains is a crucial mechanism in many physiological and pathological events. A disintegrin and metalloprotease-17 (ADAM17) is a key sheddase involved in essential processes, such as development, regeneration, and immune defense. ADAM17 exists in two conformations which differ in their disulfide connection in the membrane-proximal domain (MPD). Protein-disulfide isomerases (PDIs) on the cell surface convert the open MPD into a rigid closed form, which corresponds to inactive ADAM17. ADAM17 is expressed in its open activatable form in the endoplasmic reticulum (ER) and consequently must be protected against ER-resident PDI activity. Here, we show that the chaperone 78-kDa glucose-regulated protein (GRP78) protects the MPD against PDI-dependent disulfide-bond isomerization by binding to this domain and, thereby, preventing ADAM17 inhibition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12858DOI Listing
November 2017

Cancer-associated mutations in the canonical cleavage site do not influence CD99 shedding by the metalloprotease meprin β but alter cell migration .

Oncotarget 2017 Aug 4;8(33):54873-54888. Epub 2017 Jul 4.

Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, 24118 Kiel, Germany.

Transendothelial cell migration (TEM) is crucial for inflammation and metastasis. The adhesion molecule CD99 was shown to be important for correct immune cell extravasation and is highly expressed on certain cancer cells. Recently, we demonstrated that ectodomain shedding of CD99 by the metalloprotease meprin β promotes TEM . In this study, we employed an acute inflammation model (air pouch/carrageenan) and found significantly less infiltrated cells in meprin β knock-out animals validating the previously observed pro-inflammatory activity. To further analyze the impact of meprin β on CD99 shedding with regard to cell adhesion and proliferation we characterized two lung cancer associated CD99 variants (D92H, D92Y), carrying point mutations at the main cleavage site. Interestingly, ectodomain shedding of these variants by meprin β was still detectable. However the cleavage site shifted to adjacent positions. Nevertheless, expression of CD99 variants D92H and D92Y revealed partial misfolding and proteasomal degradation. A previously observed influence of CD99 on Src activation and increased proliferation could not be confirmed in this study, independent of wild-type CD99 or the variants D92H and D92Y. However, we identified meprin β as a potent inducer of Src phosphorylation. Importantly, we found significantly increased cell migration when expressing the cancer-associated CD99 variant D92H compared to the wild-type protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.18966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589627PMC
August 2017

A novel tertiary prep-HPLC method for the isolation of single amino acids for AMS-radiocarbon measurement.

J Chromatogr B Analyt Technol Biomed Life Sci 2017 Jul 15;1058:85-92. Epub 2017 May 15.

Leibniz-Laboratory for Radiometric Dating and Isotope Research (University of Kiel), Max-Eyth-Str. 11-13, 24118 Kiel, Germany.

AMS-radiocarbon measurements of amino acids can potentially provide more reliable radiocarbon dates than bulk collagen analysis. Nonetheless, the applicability of such an approach is often limited by the low-throughput of existing isolation methods and difficulties in determining the contamination introduced during the separation process. A novel tertiary prep-HPLC amino acid isolation method was developed that relies on the combustion of eluted material without requiring any additional chemical steps. Amino acid separation was carried out using a gradient mix of pure water and phosphoric acid with an acetonitrile step in-between runs to remove hydrophobic molecules from the separation column. The amount of contaminant carbon and its C content were determined from two-point measurements of collagen samples of known C content. The amount of foreign carbon due to the isolation process was estimated at 4±1μg and its C content was 0.43±0.01 FC. Radiocarbon values corrected for carbon contamination have only a minor increase in uncertainties. For Holocene samples, this corresponds to an added uncertainty typically smaller than 10 Cyears. The developed method can be added to routine AMS measurements without implying significant operational changes and offers a level of measurement uncertainty that is suitable for many archaeological, ecological, environmental, and biological applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jchromb.2017.05.017DOI Listing
July 2017

Meprin Metalloproteases Generate Biologically Active Soluble Interleukin-6 Receptor to Induce Trans-Signaling.

Sci Rep 2017 03 9;7:44053. Epub 2017 Mar 9.

Institute of Biochemistry, University of Kiel, 24118 Kiel, Germany.

Soluble Interleukin-6 receptor (sIL-6R) mediated trans-signaling is an important pro-inflammatory stimulus associated with pathological conditions, such as arthritis, neurodegeneration and inflammatory bowel disease. The sIL-6R is generated proteolytically from its membrane bound form and A Disintegrin And Metalloprotease (ADAM) 10 and 17 were shown to perform ectodomain shedding of the receptor in vitro and in vivo. However, under certain conditions not all sIL-6R could be assigned to ADAM10/17 activity. Here, we demonstrate that the IL-6R is a shedding substrate of soluble meprin α and membrane bound meprin β, resulting in bioactive sIL-6R that is capable of inducing IL-6 trans-signaling. We determined cleavage within the N-terminal part of the IL-6R stalk region, distinct from the cleavage site reported for ADAM10/17. Interestingly, meprin β can be shed from the cell surface by ADAM10/17 and the observation that soluble meprin β is not capable of shedding the IL-6R suggests a regulatory mechanism towards trans-signaling. Additionally, we observed a significant negative correlation of meprin β expression and IL-6R levels on human granulocytes, providing evidence for in vivo function of this proteolytic interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep44053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343444PMC
March 2017

Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT).

J Proteome Res 2017 02 7;16(2):384-392. Epub 2016 Dec 7.

Proteomics International , Perth, Western Australia 6009, Australia.

This study aimed to compare the depth and reproducibility of total proteome and differentially expressed protein coverage in technical duplicates and triplicates using iTRAQ 4-plex, iTRAQ 8-plex, and TMT 6-plex reagents. The analysis was undertaken because comprehensive comparisons of isobaric mass tag reproducibility have not been widely reported in the literature. The highest number of proteins was identified with 4-plex, followed by 8-plex and then 6-plex reagents. Quantitative analyses revealed that more differentially expressed proteins were identified with 4-plex reagents than 8-plex reagents and 6-plex reagents. Replicate reproducibility was determined to be ≥69% for technical duplicates and ≥57% for technical triplicates. The results indicate that running an 8-plex or 6-plex experiment instead of a 4-plex experiment resulted in 26 or 39% fewer protein identifications, respectively. When 4-plex spectra were searched with three software tools-ProteinPilot, Mascot, and Proteome Discoverer-the highest number of protein identifications were obtained with Mascot. The analysis of negative controls demonstrated the importance of running experiments as replicates. Overall, this study demonstrates the advantages of using iTRAQ 4-plex reagents over iTRAQ 8-plex and TMT 6-plex reagents, provides estimates of technical duplicate and triplicate reproducibility, and emphasizes the value of running replicate samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.5b01154DOI Listing
February 2017

Proteolytic Origin of the Soluble Human IL-6R In Vivo and a Decisive Role of N-Glycosylation.

PLoS Biol 2017 01 6;15(1):e2000080. Epub 2017 Jan 6.

Institute of Biochemistry, Kiel University, Kiel, Germany.

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2000080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5218472PMC
January 2017

Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration.

FASEB J 2017 03 21;31(3):1226-1237. Epub 2016 Dec 21.

Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, Kiel, Germany;

The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin β and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin β with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin β would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin β induced cell migration of Lewis lung carcinoma cells in an TEM assay. Accordingly, deficiency of meprin β in mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin β could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin β inhibitor-treated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201601113RDOI Listing
March 2017

Characterization of post-translational modifications in full-length human BMP-1 confirms the presence of a rare vicinal disulfide linkage in the catalytic domain and highlights novel features of the EGF domain.

J Proteomics 2016 Apr 2;138:136-45. Epub 2016 Mar 2.

Systematic Proteomics & Bioanalytics, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel, Germany. Electronic address:

Unlabelled: Bone morphogenetic protein 1 (BMP-1) is an essential metalloproteinase to trigger extracellular matrix assembly and organogenesis. Previous structural studies on the refolded catalytic domain of BMP-1 produced in E. coli have suggested the existence of a rare vicinal disulfide linkage near the active site. To confirm that this was not an artifact of the refolding procedure, the full-length human BMP-1 produced in mammalian cells was investigated via sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution LC-MS/MS analysis to interrogate the post-translational modifications. Ten disulfide linkages of BMP-1, including the vicinal disulfide linkage C185-C186 could be unambiguously identified. Further, around 50% of this vicinal disulfide bond was found to be modified by N-ethylmaleimide (NEM), a cysteine protease inhibitor supplied when the BMP-1-containing medium was collected, suggesting that this bond was highly unstable. In the absence of NEM, BMP-1 has a higher tendency to form aggregates, but after aggregate removal, C185 and C186 are almost quantitatively engaged in the vicinal disulfide bond and BMP-1 activity remains unchanged. In addition, three consensus N-glycosylation sites at N142, N363, and N599 could be identified together with a previously unknown O-glycosylation site and an Asn-hydroxylation.

Significance: An in-depth characterization of post-translational modifications of the full-length human BMP-1 produced in mammalian cells by MS was performed. A rare vicinal disulfide bond in the catalytic domain could be confirmed for the first time by mass spectrometry along with nine other proposed disulfide linkages of mature BMP-1. This vicinal disulfide bond can transiently open to form covalent adducts with the cysteine protease inhibitor (NEM) supplied in cell medium during protein harvesting. Further, we report a previously unknown O-glycosylation site and Asn-hydroxylation site, indicating a novel feature of BMP-1 in the EGF domain. The study clearly outlines the benefit of in-depth characterization of overexpressed proteins to deduce important protein modifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2016.02.031DOI Listing
April 2016

C-Terminal Charge-Reversal Derivatization and Parallel Use of Multiple Proteases Facilitates Identification of Protein C-Termini by C-Terminomics.

J Proteome Res 2016 Apr 17;15(4):1369-78. Epub 2016 Mar 17.

AG Systematische Proteomforschung & Bioanalytik, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel , Niemannsweg 11, 24105 Kiel, Germany.

The identification of protein C-termini in complex proteomes is challenging due to the poor ionization efficiency of the carboxyl group. Amidating the negatively charged C-termini with ethanolamine (EA) has been suggested to improve the detection of C-terminal peptides and allows for a directed depletion of internal peptides after proteolysis using carboxyl reactive polymers. In the present study, the derivatization with N,N-dimethylethylenediamine (DMEDA) and (4-aminobutyl)guanidine (AG) leading to a positively charged C-terminus was investigated. C-terminal charge-reversed peptides showed improved coverage of b- and y-ion series in the MS/MS spectra compared to their noncharged counterparts. DMEDA-derivatized peptides resulted in many peptides with charge states of 3+, which benefited from ETD fragmentation. This makes the charge-reversal strategy particularly useful for the analysis of protein C-termini, which may also be post-translationally modified. The labeling strategy and the indirect enrichment of C-termini worked with similar efficiency for both DMEDA and EA, and their applicability was demonstrated on an E. coli proteome. Utilizing two proteases and different MS/MS activation mechanisms allowed for the identification of >400 C-termini, encompassing both canonical and truncated C-termini.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.6b00146DOI Listing
April 2016

From top-down to bottom-up: Time-dependent monitoring of proteolytic protein degradation by LC-MS.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Mar 17;1015-1016:111-120. Epub 2016 Feb 17.

AG Systematische Proteomforschung & Bioanalytik, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel, Germany. Electronic address:

The understanding of proteolytic processes includes manifold aspects, ranging from the characterization of proteases and their corresponding substrates to the localization of cleavage sites. The analysis of protease-catalyzed reactions at a single time-point in many cases excludes the identification of intermediate cleavage products of potential biological function. To overcome this problem, proteolysis has to be monitored over time. For that purpose, we established a straight-forward two-step approach. First, Tricine-SDS-PAGE separation of the proteolytic products is applied to survey the proteolytic reaction. In the second step, the reaction mixture is analyzed by an LC-MS set-up. An optimized chromatographic separation coupled to electrospray Orbitrap mass spectrometry allowed the simultaneous monitoring of intact substrates, intermediates and cleavage products of lower molecular weight. The applicability of the strategy was shown on the example of the gastric protease pepsin and its physiologically relevant substrate hen egg white lysozyme, one of the major egg allergens. While lysozyme-derived cysteine-free peptides were cleaved comparatively fast, disulfide bonds protected connected peptides from rapid peptic proteolysis. Two previously identified potential IgE-binding motifs were observed as disulfide-linked cleavage products. In summary, the presented approach is not only ideally suited for the simulation of gastro-intestinal digestion, which is of high interest in food research, but can be transferred to any protease-substrate pair of interest. Furthermore the strategy can be exploited to deduce the effect of post-translational modifications on proteolysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jchromb.2016.02.021DOI Listing
March 2016

β-Lactoglobulin as nanotransporter--Part II: Characterization of the covalent protein modification by allicin and diallyl disulfide.

Food Chem 2016 Apr 4;197(Pt A):1022-9. Epub 2015 Nov 4.

Institute of Human Nutrition and Food Science, Food Technology, Christian-Albrechts-Universität zu Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany.

The whey protein β-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified β-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified β-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to β-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.foodchem.2015.11.011DOI Listing
April 2016

Characterization of an Equine α-S2-Casein Variant Due to a 1.3 kb Deletion Spanning Two Coding Exons.

PLoS One 2015 7;10(10):e0139700. Epub 2015 Oct 7.

Institute of Animal Breeding and Husbandry, Christian-Albrechts-Universität Kiel, Kiel, Germany.

The production and consumption of mare's milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows' milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplication. Findings at the DNA-level have been verified by cDNA sequencing from horse milk of mares with different genotypes. At the protein-level, we were able to show by SDS-page and in-gel digestion with subsequent LC-MS analysis that both proteins are actually expressed. The comparison with published sequences of other equids revealed that the deletion has probably occurred before the ancestor of present-day asses and zebras diverged from the horse lineage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139700PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596476PMC
June 2016

Identification and relative quantification of phosphopeptides by a combination of multi-protease digestion and isobaric labeling.

Rapid Commun Mass Spectrom 2015 May;29(10):919-26

Institut für Experimentelle Medizin - AG Systematische Proteomforschung und Bioanalytik, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany.

Rationale: The identification and the determination of the extent of protein phosphorylation are major prerequisites for the comparative analysis of this important posttranslational modification of proteins in different biological situations. High sequence coverages and the availability of straightforward quantification methods are necessary to achieve these goals.

Methods: Phosphoproteins and non-phosphorylated analogues separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were digested using four different proteases (trypsin, chymotrypsin, elastase and GluC) and the digests were isobarically labeled using eight-plex iTRAQ. The combined labeled digests were subsequently enriched using titanium dioxide and both the phosphorylated and non-phosphorylated fractions were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The influence of different data analysis algorithms (Percolator or False Discovery Rate) on the outcome of analysis was investigated.

Results: Almost complete sequence coverage could be achieved upon application of a multi-protease approach. The formation of peptides of different lengths and physicochemical properties allowed the identification and the mapping of all phosphorylation sites in the investigated model proteins. The introduction of isobaric labels allowed quantification of different peptides of the same phosphorylation site with more than one peptide, leading to significantly improved statistical confidence.

Conclusions: A workflow for the straightforward comparative analysis of protein phosphorylation in samples of low complexity, e.g. isolated proteins, was developed. The workflow is transferable to other posttranslational modifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rcm.7185DOI Listing
May 2015

Metalloprotease meprin β is activated by transmembrane serine protease matriptase-2 at the cell surface thereby enhancing APP shedding.

Biochem J 2015 Aug 15;470(1):91-103. Epub 2015 Jun 15.

Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, 24118 Kiel, Germany

Increased expression of metalloprotease meprin β is associated with fibrotic syndromes and Alzheimer's disease (AD). Hence, regulation of meprin activity might be a suitable strategy for the treatment of these conditions. Meprin β is a type 1 transmembrane protein, but can be released from the cell surface by ectodomain shedding. The protease is expressed as an inactive zymogen and requires proteolytic maturation by tryptic serine proteases. In the present study, we demonstrate, for the first time, the differences in the activation of soluble and membrane bound meprin β and suggest transmembrane serine protease 6 [TMPRSS6 or matriptase-2 (MT2)] as a new potent activator, cleaving off the propeptide of meprin β between Arg(61) and Asn(62) as determined by MS. We show that MT2, but not TMPRSS4 or pancreatic trypsin, is capable of activating full-length meprin β at the cell surface, analysed by specific fluorogenic peptide cleavage assay, Western blotting and confocal laser scanning microscopy (CLSM). Maturation of full-length meprin β is required for its activity as a cell surface sheddase, releasing the ectodomains of transmembrane proteins, as previously shown for the amyloid precursor protein (APP).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20141417DOI Listing
August 2015

Quantitative proteome profiling of CNS-infiltrating autoreactive CD4+ cells reveals selective changes during experimental autoimmune encephalomyelitis.

J Proteome Res 2014 Aug 1;13(8):3655-70. Epub 2014 Jul 1.

Chemokine Biology Laboratory, School of Molecular and Biomedical Science, University of Adelaide and Centre for Molecular Pathology , South Australia 5005, Australia.

Experimental autoimmune encephalomyelitis (EAE) is a murine model of multiple sclerosis, a chronic neurodegenerative and inflammatory autoimmune condition of the central nervous system (CNS). Pathology is driven by the infiltration of autoreactive CD4(+) lymphocytes into the CNS, where they attack neuronal sheaths causing ascending paralysis. We used an isotope-coded protein labeling approach to investigate the proteome of CD4(+) cells isolated from the spinal cord and brain of mice at various stages of EAE progression in two EAE disease models: PLP139-151-induced relapsing-remitting EAE and MOG35-55-induced chronic EAE, which emulate the two forms of human multiple sclerosis. A total of 1120 proteins were quantified across disease onset, peak-disease, and remission phases of disease, and of these 13 up-regulated proteins of interest were identified with functions relating to the regulation of inflammation, leukocyte adhesion and migration, tissue repair, and the regulation of transcription/translation. Proteins implicated in processes such as inflammation (S100A4 and S100A9) and tissue repair (annexin A1), which represent key events during EAE progression, were validated by quantitative PCR. This is the first targeted analysis of autoreactive cells purified from the CNS during EAE, highlighting fundamental CD4(+) cell-driven processes that occur during the initiation of relapse and remission stages of disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr500158rDOI Listing
August 2014

LC-MS based cleavage site profiling of the proteases ADAM10 and ADAM17 using proteome-derived peptide libraries.

J Proteome Res 2014 Apr 17;13(4):2205-14. Epub 2014 Mar 17.

AG Systematische Proteomforschung, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel , Niemannsweg 11, 24105 Kiel, Germany.

A Disintegrin and Metalloproteinase 10 (ADAM10) and ADAM17 catalyze ectodomain shedding of a number of cell surface proteins important for embryonic development and tissue homeostasis. Changes in the expression levels or dysregulated proteolytic activity of ADAM10 and ADAM17 have been shown to play important roles in multiple diseases such as inflammation, cancer, and neurodegenerative disorders. Despite the well documented substrate repertoire of ADAM10 and ADAM17, little is known about their cleavage site specificity. We optimized Q-PICS (Quantitative Proteomics for the Identification of Cleavage Sites) to elucidate the cleavage site specificity of recombinant murine ADAM10 and ADAM17. Two different yeast proteome-derived peptide libraries were used and samples were analyzed by LC-MALDI and LC-ESI MS in parallel. We show that the largest difference in the cleavage site specificities of ADAM10 and ADAM17 is at the P1' site: while both enzymes cleave N-terminal of leucine, only ADAM10 shows additional preference toward aromatic amino acids, whereas ADAM17 exhibits the highest preference for valine. Together with further amino acid preferences more adjacent to the scissile bond, our data is in good agreement with ADAM10/17 cleavage sites previously identified in native substrates. Overall, the precise identification of ADAM10 and ADAM17 cleavage site specificity provides the basis for better substrate identification in vivo and the generation of specific inhibitors or activity based probes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr401135uDOI Listing
April 2014

Mapping orphan proteases by proteomics: meprin metalloproteases deciphered as potential therapeutic targets.

Proteomics Clin Appl 2014 Jun 24;8(5-6):382-8. Epub 2014 Mar 24.

Institute of Biochemistry, Unit for Degradomics of the Protease Web, University of Kiel, Kiel, Germany.

The protease web is a synonym for highly regulated molecular networks comprising enzymes, substrates, inhibitors, and other regulatory proteins. Latest high-throughput methods provided huge data sets, revealing an amazing complexity of proteolytic systems important for health and disease. Based on our previous studies, we discuss major problems and questions that have to be solved to gain precise insight into the regulation of the protease web and its impact on pathophysiological conditions. The goal is a combination of different proteomic approaches that help to investigate specific protease function at a glance. Exemplarily, the characterization of the metalloproteases meprin α and meprin β by proteomic identification of cleavage sites and terminal amine isotopic labeling of substrates demonstrates the power of MS-based techniques. Meprins are rather orphan proteases and could not be assigned to precise biological functions until recently. Proteomics helped to identify meprin α and meprin β being important for collagen assembly and deposition in skin, which makes them potential therapeutic targets in fibrotic conditions. Additionally, identification of the cleavage site specificity provides the basis for the development of activity-based probes and small compound inhibitors, important for the regulation of meprin activity and subsequent treatment of associated diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/prca.201300079DOI Listing
June 2014

Polo-like kinase 2, a novel ADAM17 signaling component, regulates tumor necrosis factor α ectodomain shedding.

J Biol Chem 2014 Jan 13;289(5):3080-93. Epub 2013 Dec 13.

From the Institute of Biochemistry, Christian-Albrechts-Universität zu Kiel, 24118 Kiel, Germany.

ADAM17 (a disintegrin and metalloprotease 17) controls pro- and anti-inflammatory signaling events by promoting ectodomain shedding of cytokine precursors and cytokine receptors. Despite the well documented substrate repertoire of ADAM17, little is known about regulatory mechanisms, leading to substrate recognition and catalytic activation. Here we report a direct interaction of the acidophilic kinase Polo-like kinase 2 (PLK2, also known as SNK) with the cytoplasmic portion of ADAM17 through the C-terminal noncatalytic region of PLK2 containing the Polo box domains. PLK2 activity leads to ADAM17 phosphorylation at serine 794, which represents a novel phosphorylation site. Activation of ADAM17 by PLK2 results in the release of pro-TNFα and TNF receptors from the cell surface, and pharmacological inhibition of PLK2 leads to down-regulation of LPS-induced ADAM17-mediated shedding on primary macrophages and dendritic cells. Importantly, PLK2 expression is up-regulated during inflammatory conditions increasing ADAM17-mediated proteolytic events. Our findings suggest a new role for PLK2 in the regulation of inflammatory diseases by modulating ADAM17 activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.536847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908438PMC
January 2014

Metalloproteases meprin α and meprin β are C- and N-procollagen proteinases important for collagen assembly and tensile strength.

Proc Natl Acad Sci U S A 2013 Aug 12;110(35):14219-24. Epub 2013 Aug 12.

Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, 24118 Kiel, Germany.

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a(-/-) and Mep1b(-/-) mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin β are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1305464110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3761563PMC
August 2013
-->