Link peptide cartilage growth factor is degraded by membrane proteinases

2000 ◽  
Vol 349 (2) ◽  
pp. 473-479 ◽  
Author(s):  
Michael F. DEAN ◽  
Paul SANSOM
Keyword(s):  
Growth Factor ◽  
Time Course ◽  
Plasma Membranes ◽  
Serine Proteinase ◽  
Biologically Active ◽  
Cartilage Growth ◽  
Link Protein ◽  
Whole Cells ◽  
N Terminus ◽  
Model Cell

The peptide DHLSDNYTLDHDRAIH (Link N), cleaved from the N-terminus of the link protein component of cartilage proteoglycan aggregates by the action of stromelysin, can act as a growth factor and stimulate synthesis of proteoglycans and collagen in articular cartilage [McKenna, Liu, Sansom and Dean (1998) Arthritis Rheum. 41, 157-161]. The mechanism by which this biologically active peptide is degraded and inactivated was investigated using U937 monocytes as a model cell. Time-course experiments showed that two major proteases, an initial serine proteinase followed by a metalloproteinase, acted in sequence. Analysis of the resulting fragments showed that the serine endopeptidase cleavage was at the Leu3-Ser4 bond to produce the peptide SDNYTLDHDRAIH. The terminal serine could then be removed from the resulting peptide by an aminopeptidase. A second metallopeptidase liberated the peptides SDNYTL or DNYTL from DHDRAIH by cleavage at the Leu9-Asp10 bond. The DNYTL peptide intermediate was degraded too rapidly to allow sequencing and sequential aminopeptidase cleavages removed further amino acids from the N-terminus of the remaining DHDRAIH peptide. The identical patterns of breakdown that occurred when either whole cells or purified plasma membranes were used indicated that proteolysis and inactivation of Link N was carried out entirely by membrane-associated enzymes.

scholarly journals A proteolytic fragment from human link protein is taken up and processed by monocytes and B cells

1991 ◽  
Vol 280 (3) ◽  
pp. 679-686 ◽  
Author(s):  
H Martin ◽  
M Dean
Keyword(s):  
B Cells ◽  
Plasma Membranes ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Peritoneal Macrophages ◽  
High Uptake ◽  
Proteolytic Fragment ◽  
Link Protein ◽  
Protein Uptake ◽  
Proteoglycan Aggregates

Mild digestion of 125I-labelled human proteoglycan aggregates with trypsin or stromelysin produced specific peptides that were taken up rapidly by THP-1 monocytes. SDS/PAGE of undigested aggregate showed that the three components of molecular mass 48, 44 and 41 kDa, corresponding to isoforms of link protein originally present, had been converted into a single component of 41 kDa by trypsin treatment, and that fragments of 6-12 kDa were present in fractions containing the high-uptake peptide. Separate proteolysis of isolated proteoglycan monomer and link protein confirmed that the specific high-uptake fragment was derived from link protein. Uptake of the link fragment was rapid, reaching a maximum after 5 min, and specific, since it was blocked by metabolic or serine proteinase inhibitors and at 4 degrees C. After uptake the cleaved fragment was processed further, with 50% of the radiolabel being released as degraded peptides within 5 min. In contrast, accumulation of whole aggregate reached a maximum after 45 min and only 50% had been released after 2 h. Uptake of aggregate was less affected by inhibitors or at low temperature, suggesting that a separate mechanism existed for its turnover. The aggregate was transported to lysosomes after uptake, although the link fragment did not sediment with either lysosomes or plasma membranes, suggesting that it was present in the cytoplasm or in very labile vesicles. However, the mode of handling of the peptide by the cells remains unclear. The link fragment was taken up by several different monocytic and B cell lines, but not by mouse fibroblasts or peritoneal macrophages. These data suggest that a surface serine proteinase on monocytes and B cells enables them to process and take up a fragment of link protein derived by extracellular proteolysis.

scholarly journals Altering the Stereoselectivity of Whole-Cell Biotransformations via the Physicochemical Parameters Impacting the Processes

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 781
Author(s):  
Agnieszka Raczyńska ◽  
Joanna Jadczyk ◽  
Małgorzata Brzezińska-Rodak
Keyword(s):  
Culture Conditions ◽  
Biologically Active ◽  
Enantiomerically Pure ◽  
Whole Cells ◽  
Chiral Compounds ◽  
Physicochemical Factors ◽  
Pure Compounds ◽  
Genetic Level ◽  
Prochiral Ketones ◽  
Optically Pure

The enantioselective synthesis of organic compounds is one of the great challenges in organic synthetic chemistry due to its importance for the acquisition of biologically active derivatives, e.g., pharmaceuticals, agrochemicals, and others. This is why biological systems are increasingly applied as tools for chiral compounds synthesis or modification. The use of whole cells of “wild-type” microorganisms is one possible approach, especially as some methods allow improving the conversion degrees and controlling the stereoselectivity of the reaction without the need to introduce changes at the genetic level. Simple manipulation of the culture conditions, the form of a biocatalyst, or the appropriate composition of the biotransformation medium makes it possible to obtain optically pure products in a cheap, safe, and environmentally friendly manner. This review contains selected examples of the influence of physicochemical factors on the stereochemistry of the biocatalytic preparation of enantiomerically pure compounds, which is undertaken through kinetically controlled separation of their racemic mixtures or reduction of prochiral ketones and has an effect on the final enantiomeric purity and enantioselectivity of the reaction.

CLEAVAGE PATHWAY,AND SPECIFICITY OF LEUCOCYTE ELASTASE AS COMPARED TO PLASMIN DURING FIBRINOGEN DEGRADATION

1987 ◽  
Author(s):  
L Goretzki ◽  
E Miller ◽  
A Henschen
Keyword(s):  
Amino Acid ◽  
Time Course ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Reversed Phase ◽  
Cleavage Sites ◽  
Human Fibrinogen ◽  
Leucocyte Elastase ◽  
N Terminus

Plasmin and leucocyte elastase are regarded as the two medically most important fibrin(ogen)-degrading proteolytic enzymes. There is, however, a considerable difference in information available about the cleavage specificities and fragmentation pathways of these two enzymes. Degradation by plasmin has been studied already for a long time in great detail so that now the time course of the degradation, the cleavage sites and the functional properties of many fragments are well known. In contrast, relatively little is known about the degradation by leucocyte elastase, except that the overall cleavage pattern resembles that obtained with plasminIn this investigation the leucocyte elastase-mediated degradation of fibrinogen has been examined by means of proteinchemi-cal methods. Human fibrinogen was incubated with human enzyme material for various periods of time and at some different enzyme concentrations. The split products formed at the various stages were isolated in pure form by gel filtration followed by reversed-phase high-performance liquid chromatography. The fragments were identified by N-terminal amino acid sequence and amino acid composition. The course of the degradation was also monitored by sodium dodecylsulfate-polyacrylamide gel electrophoresis. All cleavage patterns were compared with the corresponding patterns from plasmic degradation. It could be confirmed that X-, D- and E-like fragments are formed also with elastase. However, several early elastolytic Aα-chain fragments are characteristically different from plasmic fragments. The previously identified N-terminal cleavage site in the Aα-chain, i.e. after position 21, was found to be the most important site in this region of fibrinogen. The very early degradation of the Aα-chain N-terminus by elastase is in strong contrast to the stability against plasmin. Several cleavage sites in N-terminal region of the Bβ-chain were observed, though the low amino acid specificity of elastase partly hampered the identification. The γ-chain N-terminus was found to be as highly stable towards elastase as towards plasmin. The results are expected to contribute to the understanding of the role of leucocyte elastase in pathophysiologic fibrino(geno)lysis

Localization of Chromogranins, Non-neuron-specific Enolase, and Different Forms of Somatostatins in the Submandibular Salivary Glands of Mice

1990 ◽  
Vol 69 (8) ◽  
pp. 1494-1499 ◽  
Author(s):  
A. Letić-Gavrilović ◽  
K. Abe
Keyword(s):  
Growth Factor ◽  
Salivary Glands ◽  
Chromogranin A ◽  
Neuron Specific Enolase ◽  
Biologically Active ◽  
Detection Methods ◽  
Duct System ◽  
Male Mice ◽  
Chromogranin B ◽  
Submandibular Salivary Glands

The localizations of chromogranins A, B, and C, neuron-specific enolase (NSE, γγ-type) and non-NSE (αα-type), and different forms of somatostatins were immunocytochemically identified. The localizations were compared with those of epidermal growth factor (EGF) and nerve growth factor (NGF) in the submandibular salivary glands (SMG) of male mice at five to six weeks of age, with use of a variety of antibodies and the peroxidase-antiperoxidase (PAP) and avidin-biotin complex (ABC) detection methods. In the SMG of male mice, the major chromogranin present was chromogranin A, whereas chromogranins B and C were not detected at these ages by either method. Chromogranin Alike immunoreactivity was located in the granular convoluted tubule (GCT) cells of the SMG, whereas non-NSE immunoreactivity was observed throughout the duct system and in some acinar-associated cells. NSE was not detected in any part of the SMG. The distribution of chromogranin A and somatostatins in the GCT cells was similar to that of EGF and NGF. Our results strongly suggest that chromogranin A and somatostatins, but not chromogranin B or C, may be useful as a means of differentiation of the cells in the duct system of the SMG responsible for the production of biologically-active factors.

scholarly journals Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

1988 ◽  
Vol 8 (3) ◽  
pp. 1247-1252 ◽  
Author(s):  
E Lazar ◽  
S Watanabe ◽  
S Dalton ◽  
M B Sporn
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Amino Acid ◽  
Growth Factor ◽  
Aspartic Acid ◽  
Transforming Growth Factor ◽  
Biologically Active ◽  
Single Amino Acid ◽  
Transforming Growth Factor Alpha ◽  
Factor Alpha

To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.

scholarly journals Acetylcholine Receptors

1972 ◽  
Vol 60 (3) ◽  
pp. 248-262 ◽  
Author(s):  
H. Criss Hartzell ◽  
Douglas M. Fambrough
Keyword(s):  
Acetylcholine Receptors ◽  
Time Course ◽  
Plasma Membranes ◽  
Scintillation Counting ◽  
Receptor Density ◽  
Rat Diaphragm ◽  
Leg Muscles ◽  
And Function ◽  
Quantitative Radioautography ◽  
Ach Receptors

Using 125iodine-labeled α-bungarotoxin (α-BGT-125I) and quantitative radioautography, we have studied the time-course of the change in acetylcholine (ACh) receptor distribution and density occurring in rat diaphragm after denervation. In innervated fibers, ACh receptors are localized at the neuromuscular junction and the extrajunctional receptor density is less than five receptors per square micrometer. The extrajunctional receptor density begins to increase between 2 and 3 days after denervation and increases approximately linearly to 1695 receptors/µm2 at 14 days, subsequently decreasing to 529 receptors/µm2 at 45 days. We have isolated plasma membranes from rat leg muscles at various times after denervation and find that the change in concentration of ACh receptors in the membranes measured by α-BGT-125I binding and scintillation counting follows a time-course similar to the change in ACh receptor density measured radioautographically. Furthermore, we have correlated extrajunctional ACh receptor density measured by radioautography with extrajunctional ACh sensitivity measured by iontophoretic application of ACh and intracellular recording and find that the log of ACh receptor density is related to 0.53 times the log of ACh sensitivity. These results are discussed in terms of the electrophysiological experiments on the ACh receptor and the recent, more biochemical approaches to the study of ACh receptor control and function.

scholarly journals Highly Sulfated Nonreducing End-derived Heparan Sulfate Domains Bind Fibroblast Growth Factor-2 with High Affinity and Are Enriched in Biologically Active Fractions

2011 ◽  
Vol 286 (22) ◽  
pp. 19311-19319 ◽  
Author(s):  
Hicham Naimy ◽  
Jo Ann Buczek-Thomas ◽  
Matthew A. Nugent ◽  
Nancy Leymarie ◽  
Joseph Zaia
Keyword(s):  
Growth Factor ◽  
Heparan Sulfate ◽  
Fibroblast Growth Factor ◽  
Ternary Complex ◽  
Biologically Active ◽  
Fibroblast Growth Factor 2 ◽  
Fibroblast Growth ◽  
High Affinity ◽  
Symmetrical Model ◽  
Asymmetrical Model

Human fibroblast growth factor-2 (FGF2) regulates cellular processes including proliferation, adhesion, motility, and angiogenesis. FGF2 exerts its biological function by binding and dimerizing its receptor (FGFR), which activates signal transduction cascades. Effective binding of FGF2 to its receptor requires the presence of heparan sulfate (HS), a linear polysaccharide with N-sulfated domains (NS) localized at the cell surface and extracellular matrix. HS acts as a platform facilitating the formation of a functional FGF-FGFR-HS ternary complex. Crystal structures of the signaling ternary complex revealed two conflicting architectures. In the asymmetrical model, two FGFs and two FGFRs bind a single HS chain. In contrast, the symmetrical model postulates that one FGF and one FGFR bind to the free end of the HS chain and dimerization require these ends to join, bringing the two half-complexes together. In this study, we screened a hexasaccharide HS library for compositions that are able to bind FGF2. The library was composed primarily of NS domains internal to the HS chain with minor presence of non-reducing end (NRE) NS. The binders were categorized into low versus high affinity binders. The low affinity fraction contained primarily hexasaccharides with low degree of sulfation that were internal to the HS chains. In contrast, the high affinity bound fraction was enriched in NRE oligosaccharides that were considerably more sulfated and had the ability to promote FGFR-mediated cell proliferation. The results suggest a role of the NRE of HS in FGF2 signaling and favor the formation of the symmetrical architecture on short NS domains.

scholarly journals Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils

1996 ◽  
Vol 314 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R. Alexander BLACKWOOD ◽  
James E. SMOLEN ◽  
Ronald J. HESSLER ◽  
Donna M. HARSH ◽  
Amy TRANSUE
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Phospholipid Vesicle ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Fluorescent Marker ◽  
Whole Cells ◽  
Specific Granules ◽  
Neutrophil Degranulation

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

scholarly journals THE “CARTILAGE GROWTH FACTOR” AND VITAMIN B6 IN THE NUTRITION OF CHICKS

1939 ◽  
Vol 130 (1) ◽  
pp. 423-424
Author(s):  
D. Mark Hegsted ◽  
J.J. Oleson ◽  
C.A. Elvehjem ◽  
E.B. Hart
Keyword(s):  
Growth Factor ◽  
Vitamin B6 ◽  
Cartilage Growth
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