scholarly journals Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis

1995 ◽  
Vol 312 (1) ◽  
pp. 107-114 ◽  
Author(s):  
P H C van Berkel ◽  
M E J Geerts ◽  
H A van Veen ◽  
P M Kooiman ◽  
F R Pieper ◽  
...  
Keyword(s):  
Human Kidney ◽  
Exchange Chromatography ◽  
Protein Sequencing ◽  
Bacterial Lipopolysaccharide ◽  
Terminal Sequence ◽  
Human Lysozyme ◽  
Major Function ◽  
Sds Page ◽  
Anti Inflammatory

We studied the role of N-glycosylation of human lactoferrin (hLF) with respect to properties that are relevant to its antibacterial and anti-inflammatory activities. A human kidney-derived 293(S) cell line that constitutively expresses recombinant hLF (rhLF) was produced. The reactivity towards various antibodies of rhLF that had been expressed in the absence or presence of tunicamycin (which blocks N-linked glycosylation) did not differ from that of natural (human milk-derived) hLF. Cation-exchange chromatography and N-terminal protein sequencing showed identical cationic properties and an intact N-terminal sequence for rhLF and natural hLF. SDS/PAGE of rhLF expressed in the presence of tunicamycin revealed a protein with the same M(r) as that of enzymically deglycosylated natural hLF. Both glycosylated and unglycosylated rhLF appeared to be completely saturated with iron. The affinity of natural hLF, glycosylated and non-glycosylated rhLF for both human lysozyme (Kd 4.5 x 10(-8) M) and bacterial lipopolysaccharide did not differ. SDS/PAGE of hLF species subjected to trypsin indicated that unglycosylated rhLF was much more susceptible to degradation. Furthermore, this analysis suggests that N-glycosylation heterogeneity in natural hLF and rhLF resides in the C-lobe. Thus our results provide no argument for differential antibacterial and/or anti-inflammatory activity of natural and (glycosylated) rhLF and suggest that a major function of glycosylation in hLF is to protect it against proteolysis.

scholarly journals N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA

1997 ◽  
Vol 328 (1) ◽  
pp. 145-151 ◽  
Author(s):  
Patrick H. C. VAN BERKEL ◽  
E. J. Marlieke GEERTS ◽  
A. Harry VAN VEEN ◽  
Mathias MERICSKAY ◽  
A. Herman DE BOER ◽  
...  
Keyword(s):  
Lipid A ◽  
Solid Phase ◽  
Decisive Role ◽  
Bacterial Lipopolysaccharide ◽  
Human Lactoferrin ◽  
Binding Assays ◽  
Human Lysozyme ◽  
Ligand Interaction ◽  
Arginine Residues

Human lactoferrin (hLF), a protein involved in host defence against infection and excessive inflammation, interacts with heparin, the lipid A moiety of bacterial lipopolysaccharide, human lysozyme (hLZ) and DNA. To determine which region of the molecule is important in these interactions, solid-phase ligand binding assays were performed with hLF from human milk (natural hLF) and N-terminally deleted hLF variants. Iron-saturated and natural hLF bound equally well to heparin, lipid A, hLZ and DNA. Natural hLF lacking the first two N-terminal amino acids (Gly1-Arg2) showed reactivities of one-half, two-thirds, one-third and one-third towards heparin, lipid A, hLZ and DNA respectively compared with N-terminally intact hLF. A lack of the first three residues (Gly1-Arg2-Arg3) decreased binding to the same ligands to one-eighth, one-quarter, one-twentieth and one-seventeenth respectively. No binding occurred with a mutant lacking the first five residues (Gly1-Arg2-Arg3-Arg4-Arg5). An anti-hLF monoclonal antibody (E11) that reacts to an N-lobe epitope including Arg5 completely blocked hLF-ligand interaction. These results show that the N-terminal stretch of four consecutive arginine residues, Arg2-Arg3-Arg4-Arg5, has a decisive role in the interaction of hLF with heparin, lipid A, hLZ and DNA. The role of limited N-terminal proteolysis of hLF in its anti-infective and anti-inflammatory properties is discussed.

scholarly journals A novel neutrophil chemoattractant generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to interleukin 8

1990 ◽  
Vol 271 (3) ◽  
pp. 797-801 ◽  
Author(s):  
B C Beaubien ◽  
P D Collins ◽  
P J Jose ◽  
N F Totty ◽  
J Hsuan ◽  
...  
Keyword(s):  
Peritoneal Cavity ◽  
Inflammatory Reaction ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Interleukin 8 ◽  
Terminal Sequence ◽  
Rabbit Skin ◽  
Sds Page

An inflammatory reaction was induced in vivo by injection of zymosan into the peritoneal cavity of the rabbit. The inflammatory exudate was found to contain oedema-inducing and neutrophil chemoattractant activity when assayed in rabbit skin in vivo, using 125I-albumin and 111In-neutrophils. This activity was additional to that of complement fragment C5a, which was removed by an affinity gel. Two chemoattractants were isolated by cation-exchange, gel-filtration and reversed-phase h.p.l.c. One of these, which ran as a single band of 6-8 kDa on SDS/PAGE, was subjected to N-terminal sequence analysis without reduction and alkylation of cysteine residues. Positive identification of 28 of the first 31 amino acids revealed a rabbit homologue of interleukin-8 (75% sequence identity with human interleukin-8). The demonstration of interleukin-8 as a major neutrophil chemoattractant in an inflammatory reaction in vivo provides the basis for further investigations into the role of this cytokine in the inflammatory process.

scholarly journals Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 561-569 ◽  
Author(s):  
Sadao Manabe ◽  
Hirofumi Nariya ◽  
Shigeru Miyata ◽  
Hiroaki Tanaka ◽  
Junzaburo Minami ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Biological Properties ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Light Chains ◽  
Sds Page ◽  
The Difference ◽  
Culture Supernatants

Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (Clp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38 kDa) and light (16 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity toward N-benzoyl-l-arginine p-nitroanilide (Bz-l-arginine pNA), as demonstrated by assaying culture supernatants differing in the relative ratio of the two light chains. Although the purified Clp preferentially degraded Bz-dl-arginine pNA rather than Bz-dl-lysine pNA, it degraded the latter more efficiently than did Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. Clp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis.

Fibrino(geno)lytic Properties of Purified Hementerin, a Metalloproteinase from the Leech Haementeria depressa

1998 ◽  
Vol 80 (07) ◽  
pp. 155-160 ◽  
Author(s):  
Ana Marisa Chudzinski-Tavassi ◽  
Eva Maria Kelen ◽  
Ana Paula de Paula Rosa ◽  
Stephane Loyau ◽  
Claudio Sampaio ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Exchange Chromatography ◽  
Single Chain ◽  
Terminal Sequence ◽  
Calcium Dependent ◽  
Amino Terminal ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Amino Terminal Sequence

SummaryThe fibrino(geno)lytic protein designated hementerin contained in crude extracts of the salivary complex of Haementeria depressa leeches was purified to apparent homogeneity by gel filtration, ion exchange chromatography and preparative SDS-PAGE. It is a single-chain 80 kDa, PhMeSO2F-resistant, calcium-dependent, metalloproteinase, which specifically degrades fibrin(ogen) through a plasminogen-independent pathway. The amino terminal sequence of 8 residues shows 80% similarity with hementin, another fibrino(geno)lytic protein purified from Haementeria ghilianii leeches. However, their activities differ somewhat in terms of kinetics and with regard to the structure of the fibrin(ogen) fragments they may produce. Cleavage by hementerin of fibrinogen Aα, γ and Bβ chains, in that order, produces 270 kDa to 67 kDa fragments which differ from those produced by plasmin. Hementerin was also able to degrade cross-linked fibrin although at a lower rate as compared to fibrinogen. In conclusion, hementerin is a plasminogen-independent fibrino(geno)lytic metalloproteinase that degrades fibrinogen faster than fibrin, prevents blood coagulation and destroys fibrin clots in vitro.

scholarly journals The role of C4-binding protein and {beta }1H in proteolysis of C4b and C3b

1979 ◽  
Vol 150 (2) ◽  
pp. 267-276 ◽  
Author(s):  
T Fujita ◽  
V Nussenzweig
Keyword(s):  
Binding Protein ◽  
Fluid Phase ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Net Charge ◽  
Molecular Weights ◽  
Physico Chemical ◽  
Sds Page ◽  
A Chain

Two forms of C4-binding protein (C4-bp) (C4-bp low, C4-bp high), which differ slightly in net charge and apparent molecular weight, as determined by SDS- PAGE, were separated by ion-exchange chromatography and contaminants removed with specific antisera. Both forms of C4-bp served as cofactors for the cleavage of C4b in solution by C3b inactivator, and the resulting fragments of the a'-chain of C4b had identical molecular weights. In addition, similarly to β1H, C4-bp low or high served as cofactors for the cleavage of fluid phase C3b by C3bINA. However, important quantitative differences between the activities of C4-bp and β1H were observed. With regard to C3b in solution, the cofactor activity of β1H was {approximately equal to}20 times greater than that of C4-bp on a weight basis. In relation to cell-bound C3b, the differences in activity were even more marked. Whereas β1H enhanced the effects of C3bINA on the erythrocyte intermediate EC3b, inhibiting the assembly of EC3bBb, C4-bp was without effect even at concentrations {approximately equal to}300 times greater than β1H. Therefore, under physiological conditions, it is likely that β1H is the key protein which controls the function of C3b, and that C4-bp activity is directed mainly toward the cleavage of C4b. We also examined the relation between C4-bp and the C3b-C4bINA cofactor described by Stroud and collaborators (3, 4). By functional, physico-chemical and immunological criteria, they are the same protein.

ANTITHROMBIN III NORTHWICK PARK: CHARACTERIZATION OF AN INACTIVE HIGH MW COMPLEX WITH INCREASED AFFINITY FOR HEPARIN

1987 ◽  
Author(s):  
H Erdjument ◽  
D A Lane ◽  
A M Flynn ◽  
H Ireland ◽  
M Panico ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Exchange Chromatography ◽  
Terminal Sequence ◽  
Structural Investigations ◽  
Crossed Immunoelectrophoresis ◽  
Sds Page ◽  
Affected Family Member ◽  
At Iii ◽  
Salt Gradient

It has been shown previously that antithrombin III Northwick Park (AT III NWP) has reduced ability to inactivate thrombin and is characterised by an additional anodal component on crossed immunoelectrophoresis. We have applied plasma from an affected family member to heparin-Sepharose and eluted the AT III with a salt gradient. Evidence will be presented that the anodal component has* higher affinity for heparin than normal AT III. Furthermore, this variant component is present in plasma as a MW >120,000 inactive complex whose tryptic peptide FAB map contains numerous signals not characteristic of normal AT . 111 . This complex can be reduced with dithiothreitol to two non identical bands on SDS PAGE with MW ~60,000, only one of which reacts with anti-AT III. Using ion-exchange chromatography and HPLC these two components have been isolated and separated. The N-terminal sequence of the protein that does not react with anti-AT III is believed to be Asp-Ala-His-Ile-Ser-Glu. Structural investigations on the variant AT III are underway.

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