Purification and characterization of a rabbit bone metalloproteinase that degrades proteoglycan and other connective-tissue components

A metalloproteinase, ‘proteoglycanase’, that degrades proteoglycan and insoluble type IV collagen as well as casein was purified to homogeneity from rabbit bone culture medium. The major form of this proteinase had a final specific activity of 2400 micrograms of casein degraded/min per mg of enzyme protein, and Mr 24 500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or 12 500 by gel-filtration chromatography. It was active over the pH range 5.0-9.0 against a number of substrates, and the rates of degradation were almost constant over the whole of this range. The products generated from proteoglycan-aggregate degradation by this enzyme indicated cleavage at multiple chondroitin sulphate-binding sites along the protein core. In a new assay to detect degradation of insoluble type IV collagen, the proteoglycanase generated large fragments, probably by cleavage in the non-helical regions. The enzyme degraded laminin, fibronectin and procollagen, removing the extension peptides of the last-mentioned. It also cleaved the ‘weak region’ of the type III collagen helix in a manner analogous to trypsin. The synthetic substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 was cleaved exclusively at the Gly-Ile bond. The proteoglycanase was inhibited by tissue inhibitors of metalloproteinases from rabbit bone culture medium, human amniotic fluid and bovine nasal-cartilage extracts, forming essentially irreversible inactive complexes. The importance of this tissue-derived enzyme, with such a wide-ranging degradative capacity, in normal and pathological connective-tissue matrix degradation is discussed.

Download Full-text

Characterization of muscle epimysium, perimysium and endomysium collagens

Biochemical Journal ◽

10.1042/bj2191017 ◽

1984 ◽

Vol 219 (3) ◽

pp. 1017-1026 ◽

Cited By ~ 141

Author(s):

N Light ◽

A E Champion

Keyword(s):

Connective Tissue ◽

Sodium Dodecyl Sulphate ◽

Type Iv Collagen ◽

Sodium Dodecyl ◽

Basement Membranes ◽

Type Iii Collagen ◽

Type I ◽

Type V Collagen ◽

Type Iv ◽

Type V

In the past it has been proven difficult to separate and characterize collagen from muscle because of its relative paucity in this tissue. The present report presents a comprehensive methodology, combining methods previously described by McCollester [(1962) Biochim. Biophys. Acta 57, 427-437] and Laurent, Cockerill, McAnulty & Hastings [(1981) Anal. Biochem. 113, 301-312], in which the three major tracts of muscle connective tissue, the epimysium, perimysium and endomysium, may be prepared and separated from the bulk of muscle protein. Connective tissue thus prepared may be washed with salt and treated with pepsin to liberate soluble native collagen, or can be washed with sodium dodecyl sulphate to produce a very clean insoluble collagenous product. This latter type of preparation may be used for quantification of the ratio of the major genetic forms of collagen or for measurement of reducible cross-link content to give reproducible results. It was shown that both the epimysium and perimysium contain type I collagen as the major component and type III collagen as a minor component; perimysium also contained traces of type V collagen. The endomysium, the sheaths of individual muscle fibres, was shown to contain both type I and type III collagen as major components. Type V collagen was also present in small amounts, and type IV collagen, the collagenous component of basement membranes, was purified from endomysial preparations. This is the first biochemical demonstration of the presence of type IV collagen in muscle endomysium. The preparation was shown to be very similar to other type IV collagens from other basement membranes on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and was indistinguishable from EHS sarcoma collagen and placenta type IV collagen in the electron microscope after rotary shadowing.

Download Full-text

Metalloproteinases from rabbit bone culture medium degrade types IV and V collagens, laminin and fibronectin

Biochemical Journal ◽

10.1042/bj1990807 ◽

1981 ◽

Vol 199 (3) ◽

pp. 807-811 ◽

Cited By ~ 92

Author(s):

G Murphy ◽

T E Cawston ◽

W A Galloway ◽

M J Barnes ◽

R A D Bunning ◽

...

Keyword(s):

Molecular Weight ◽

Culture Medium ◽

Type Iv Collagen ◽

Gel Filtration ◽

Lower Molecular Weight ◽

Bone Culture ◽

Gel Filtration Chromatography ◽

Type Iv ◽

Rabbit Bone ◽

Type V

Gel-filtration chromatography of culture medium from rabbit bone explants separates three latent metalloproteinases with activities against collagen, proteoglycan and gelatin respectively. The fractions degrading proteoglycan also degrade laminin, fibronectin and the polymeric products of pepsin-solubilized type IV collagen and can also solubilize insoluble type IV collagen. The fractions degrading gelatin are capable of degrading solubilized type V and 1 alpha,2 alpha,3 alpha (cartilage) collagens, as well as the lower-molecular-weight products of pepsin-solubilized type IV collagen. All activities can be inhibited by 1,10-phenanthroline and occur in either partially or totally latent forms that can be activated by 4-aminophenylmercuric acetate.

Download Full-text

Hereditary Connective Tissue Diseases in Young Adult Stroke: A Comprehensive Synthesis

Stroke Research and Treatment ◽

10.4061/2011/712903 ◽

2011 ◽

Vol 2011 ◽

pp. 1-18 ◽

Cited By ~ 4

Author(s):

Olivier M. Vanakker ◽

Dimitri Hemelsoet ◽

Anne De Paepe

Keyword(s):

Connective Tissue ◽

Metabolic Disorders ◽

Type Iv Collagen ◽

Connective Tissue Diseases ◽

Haemorrhagic Stroke ◽

Monogenic Disease ◽

Connective Tissue Disorders ◽

Ehlers Danlos Syndrome ◽

Type Iv ◽

Danlos Syndrome

Though the genetic background of ischaemic and haemorrhagic stroke is often polygenetic or multifactorial, it can in some cases result from a monogenic disease, particularly in young adults. Besides arteriopathies and metabolic disorders, several connective tissue diseases can present with stroke. While some of these diseases have been recognized for decades as causes of stroke, such as the vascular Ehlers-Danlos syndrome, others only recently came to attention as being involved in stroke pathogenesis, such as those related to Type IV collagen. This paper discusses each of these connective tissue disorders and their relation with stroke briefly, emphasizing the main clinical features which can lead to their diagnosis.

Download Full-text

Expression of Type IV Collagen-degrading Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Newly Established Human Oral Malignant Tumor Lines

Japanese Journal of Clinical Oncology ◽

10.1093/oxfordjournals.jjco.a023193 ◽

1996 ◽

Vol 26 (3) ◽

pp. 117-123 ◽

Cited By ~ 3

Author(s):

M. Nii ◽

Y. Kayada ◽

K. Yoshiga ◽

K. Takada ◽

K. Yanagihara

Keyword(s):

Malignant Tumor ◽

Type Iv Collagen ◽

Tissue Inhibitors Of Metalloproteinases ◽

Tissue Inhibitors ◽

Type Iv

Download Full-text

Procollagenase activator produced by rabbit uterine cervical fibroblasts

Biochemical Journal ◽

10.1042/bj2410527 ◽

1987 ◽

Vol 241 (2) ◽

pp. 527-534 ◽

Cited By ~ 17

Author(s):

M Ishibashi ◽

A Ito ◽

K Sakyo ◽

Y Mori

Keyword(s):

Culture Medium ◽

Proteinase Inhibitors ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Limited Proteolysis ◽

Serine Proteinase ◽

Active Species ◽

Rabbit Plasma ◽

Neutral Proteinase ◽

Type Iv

Culture medium from rabbit uterine cervical fibroblasts contained a procollagenase and a neutral proproteinase which acts as a procollagenase activator. These two proenzymes have been purified by a combination of ion-exchange, affinity and gel chromatographies. The purified neutral proproteinase showed Mr 60,000 with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This neutral proproteinase was activated by trypsin, 4-aminophenylmercuric acetate (APMA) and plasmin, and the active species of the proteinase had Mr 53,000 when activated by APMA; kallikrein and urokinase did not activate this proproteinase. The purified neutral proteinase was inhibited by EDTA, 1,10-phenanthroline and rabbit plasma, but not by serine proteinase inhibitors, suggesting that this proteinase is a metal-dependent proteinase. The purified enzyme could also degrade gelatin, casein, proteoglycan and type IV procollagen. The purified procollagenase had Mr 55,000 and was activated by trypsin, APMA and the active neutral proteinase. These activations were accompanied by decrease in Mr, and the activated species had an Mr which was approx. 10,000 less than that of the procollagenase. In particular, procollagenase activation with neutral proteinase depended on incubation time and proteolytic activity of proteinase. These results indicate that activation of procollagenase by the rabbit uterine neutral proteinase is related to limited proteolysis in the procollagenase molecule.

Download Full-text

An Ehrlich chromogen in collagen cross-links

Biochemical Journal ◽

10.1042/bj2090263 ◽

1983 ◽

Vol 209 (1) ◽

pp. 263-264 ◽

Cited By ~ 25

Author(s):

J E Scott ◽

R G Qian ◽

W Henkel ◽

R W Glanville

Keyword(s):

Type Iv Collagen ◽

Rich Source ◽

Type Iii Collagen ◽

Cross Link ◽

Type Iii ◽

Human Type ◽

Type Iv ◽

Cross Links

A well-characterized three-chain peptide [(Col1)2 X T9] from human type III collagen was a rich source of Ehrlich chromogen. The corresponding two-chain peptide [(Col1)2] was not, implying that the Ehrlich chromogen is a trifunctional cross-link. (Col1)2 X T9 also contained pyridinoline, which is not an Ehrlich chromogen. The 7S domain of type IV collagen also contained an Ehrlich chromogen.

Download Full-text

Modulation of extracellular matrix biosynthesis by bovine retinal pericytes in vitro: effects of the substratum and cell density

Journal of Cell Science ◽

10.1242/jcs.96.1.159 ◽

1990 ◽

Vol 96 (1) ◽

pp. 159-169

Author(s):

A.E. Canfield ◽

T.D. Allen ◽

M.E. Grant ◽

S.L. Schor ◽

A.M. Schor

Keyword(s):

Extracellular Matrix ◽

Type Iv Collagen ◽

Type I Collagen ◽

Single Cells ◽

Collagen Gel ◽

Type Iii Collagen ◽

Type I ◽

Experimental Conditions ◽

Type Iv ◽

Retinal Pericytes

Bovine retinal pericytes plated on a two-dimensional substratum display a characteristic stellate morphology. In post-confluent cultures these cells aggregate spontaneously to form multicellular nodules. The same cells plated within a three-dimensional collagen matrix display an elongated sprouting morphology. Sprouting pericytes may be embedded within a gel either as individual cells or as multicellular aggregates. We have compared the nature of the matrix proteins synthesised by pericytes displaying these different phenotypes. Stellate pericytes cultured on plastic dishes synthesised predominantly type I collagen, some type III collagen and only traces of type IV collagen. The same collagen types were secreted when nodules had formed in postconfluent cultures on plastic, and by sprouting cells plated as single cells within the collagen gel. By contrast, sprouting pericytes plated as aggregates within the collagen gel secreted increased levels of type IV collagen and reduced amounts of type I collagen. Fibronectin was synthesized by pericytes under all experimental conditions examined; thrombospondin was produced in relatively large amounts by cells grown on plastic dishes, whereas only trace amounts could be detected in the medium when the cells were cultured within a collagen gel matrix. Transmission electron microscopy revealed that pericyte aggregates within a collagen gel contained cells in close apposition surrounded by a dense extracellular matrix. In contrast, cells in the centre of a nodule on plastic appeared to be separated from each other by loose extracellular material. These results suggest that the morphological and biosynthetic phenotypes of retinal pericytes are modulated by cell-matrix and/or cell-cell interactions.

Download Full-text

Distribution of laminin and collagens during avian neural crest development

Development ◽

10.1242/dev.101.3.461 ◽

1987 ◽

Vol 101 (3) ◽

pp. 461-478 ◽

Cited By ~ 1

Author(s):

J.L. Duband ◽

J.P. Thiery

Keyword(s):

Cell Migration ◽

Neural Crest ◽

Type Iv Collagen ◽

Type I Collagen ◽

Neural Crest Cell ◽

Type Iii Collagen ◽

Type I ◽

Type Iii ◽

Type Iv ◽

Crest Cell

The distribution of type I, III and IV collagens and laminin during neural crest development was studied by immunofluorescence labelling of early avian embryos. These components, except type III collagen, were present prior to both cephalic and trunk neural crest appearance. Type I collagen was widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin shared a common distribution primarily in the basal surfaces of epithelia and in close association with developing nerves and muscle. In striking contrast with the other collagens and laminin, type III collagen appeared secondarily during embryogenesis in a restricted pattern in connective tissues. The distribution and fate of laminin and type I and IV collagens could be correlated spatially and temporally with morphogenetic events during neural crest development. Type IV collagen and lamin disappeared from the basal surface of the neural tube at sites where neural crest cells were emerging. During the course of neural crest cell migration, type I collagen was particularly abundant along migratory pathways whereas type IV collagen and laminin were distributed in the basal surfaces of the epithelia lining these pathways but were rarely seen in large amounts among neural crest cells. In contrast, termination of neural crest cell migration and aggregation into ganglia were correlated in many cases with the loss of type I collagen and with the appearance of type IV collagen and laminin among the neural crest population. Type III collagen was not observed associated with neural crest cells during their development. These observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities during neural crest ontogeny. (i) Type I collagen associated with fibronectins is a major component of the extracellular spaces of the young embryo. Together with other components, it may contribute to the three-dimensional organization and functions of the matrix during neural crest cell migration. (ii) Type III collagen is apparently not required for tissue remodelling and cell migration during early embryogenesis. (iii) Type IV collagen and laminin are important components of the basal surface of epithelia and their distribution is consistent with tissue remodelling that occurs during neural crest cell emigration and aggregation into ganglia.

Download Full-text

Blood-based extracellular matrix biomarkers are correlated with clinical outcome after PD-1 inhibition in patients with metastatic melanoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001193 ◽

2020 ◽

Vol 8 (2) ◽

pp. e001193

Author(s):

Daan P Hurkmans ◽

Christina Jensen ◽

Stijn L W Koolen ◽

Joachim Aerts ◽

Morten Asser Karsdal ◽

...

Keyword(s):

Extracellular Matrix ◽

Clinical Outcome ◽

Metastatic Melanoma ◽

Type Iv Collagen ◽

Type Iii Collagen ◽

Cox Regression Analysis ◽

High Baseline ◽

Type Iii ◽

Type Iv ◽

M1 Macrophage

BackgroundImmune checkpoint inhibitors that target the programmed cell death protein 1 (PD-1) receptor induce a response in only a subgroup of patients with metastatic melanoma. Previous research suggests that transforming growth factor beta signaling and a collagen-rich peritumoral stroma (tumor fibrosis), may negatively interfere with the interaction between T cells and tumor cells and thereby contribute to resistance mechanisms by immune-exclusion, while increased tumor infiltration of M1-like macrophages enhances T cell activity. Hence, the current study aimed to assess the relationship between blood-based markers of collagen or vimentin turnover (reflecting M1 macrophage activity) and clinical outcome in patients with metastatic melanoma after PD-1 inhibition.MethodsPatients with metastatic melanoma who were treated with anti-PD-1 monotherapy between May 2016 and March 2019 were included in a prospective observational study. N-terminal pro-peptide of type III collagen (PRO-C3) cross-linked N-terminal pro-peptides of type III collagen (PC3X), matrix metalloprotease (MMP)-degraded type III (C3M) and type IV collagen (C4M), granzyme B-degraded type IV collagen and citrullinated and MMP-degraded vimentin (VICM) were measured with immunoassays in serum before (n=107), and 6 weeks after the first administration of immunotherapy (n=94). The association between biomarker levels and overall survival (OS) or progression-free survival (PFS) was assessed.ResultsMultivariate Cox regression analysis identified high baseline PRO-C3 (Q4) and PC3X (Q4) as independent variables of worse PFS (PRO-C3: HR=1.81, 95% CI=1.06 to 3.10, p=0.030 and PC3X: HR=1.86, 95% CI=1.09 to 3.18, p=0.023). High baseline PRO-C3 was also independently related to worse OS (HR=2.08, 95% CI=1.06 to 4.09, p=0.035), whereas a high C3M/PRO-C3 ratio was related to improved OS (HR=0.42, 95% CI=0.20 to 0.90, p=0.025). An increase in VICM (p<0.0001; in 56% of the patients) was observed after 6 weeks of treatment, and an increase in VICM was independently associated with improved OS (HR=0.28, 95% CI=0.10 to 0.77, p=0.014).ConclusionsBlood-based biomarkers reflecting excessive type III collagen turnover were associated with worse OS and PFS after PD-1 inhibition in metastatic melanoma. Moreover, an increase in VICM levels after 6 weeks of treatment was associated with improved OS. These findings suggest that type III collagen and vimentin turnover contribute to resistance/response mechanisms of PD-1 inhibitors and hold promise of assessing extracellular matrix-derived and stroma-derived components to predict immunotherapy response.

Download Full-text