The role of N‐linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis

Veen, Harrie A. van; Geerts, M.E.J.; Berkel, Patrick H.C. van; Nuijens, Jan H.

doi:10.1111/j.1432-1033.2003.03965.x

Cited by 112 publications

(72 citation statements)

References 24 publications

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“…After 3 h of incubation of LF with P. gingivalis whole cells in a physiological buffer, only two major polypeptides (33 and 53 kDa) were detected, and these fragments resulted from cleavage at a single site. LF has been reported to be relatively resistant to degradation by both trypsin and chymotrypsin, and the N-linked glycosylation of LF has been shown to help protect the protein from trypsin hydrolysis (7,53). In our study, LF was more extensively hydrolyzed by trypsin than the P. gingivalis proteinases (Fig.…”

Section: Discussionmentioning

confidence: 48%

Lactoferrin Inhibits Porphyromonas gingivalis Proteinases and Has Sustained Biofilm Inhibitory Activity

Dashper

Pan

Veith

et al. 2012

Antimicrob Agents Chemother

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Porphyromonas gingivalis is a bacterial pathogen associated with chronic periodontitis that results in destruction of the tooth's supporting tissues. The major virulence determinants of P. gingivalis are its cell surface Arg-and Lys-specific cysteine proteinases, RgpA/B and Kgp. Lactoferrin (LF), an 80-kDa iron-binding glycoprotein found in saliva and gingival crevicular fluid, is believed to play an important role in innate immunity. In this study, bovine milk LF displayed proteinase inhibitory activity against P. gingivalis whole cells, significantly inhibiting both Arg-and Lys-specific proteolytic activities. LF inhibited the Argspecific activity of purified RgpB, which lacks adhesin domains, and also inhibited the same activity of the RgpA/Kgp proteinaseadhesin complexes in a time-dependent manner, with a first-order inactivation rate constant (k inact ) of 0.023 min ؊1 and an inhibitor affinity constant (K I ) of 5.02 M. LF inhibited P. gingivalis biofilm formation by >80% at concentrations above 0.625 M. LF was relatively resistant to hydrolysis by P. gingivalis cells but was cleaved into two major polypeptides (53 and 33 kDa) at R 284 to S 285 , as determined by in-source decay mass spectrometry; however, these polypeptides remained associated with each other and retained inhibitory activity. The biofilm inhibitory activity of LF against P. gingivalis was not attributed to direct antibacterial activity, as LF displayed little growth inhibitory activity against planktonic cells. As the known RgpA/B and Kgp inhibitor N-␣-p-tosyl-L-lysine chloromethylketone also inhibited P. gingivalis biofilm formation, the antibiofilm effect of LF may at least in part be attributable to its antiproteinase activity.

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Section: Discussionmentioning

confidence: 48%

Lactoferrin Inhibits Porphyromonas gingivalis Proteinases and Has Sustained Biofilm Inhibitory Activity

Dashper

Pan

Veith

et al. 2012

Antimicrob Agents Chemother

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“…Briefly, a genomic hLF sequence with polymorphic amino acids at position 4 (insertion of Arg), 11 (Ala), 29 (Arg) and 561 (Asp) (van Veen et al, 2004) under control of regulatory elements from the bovine aS 1 casein gene, was introduced into the bovine germline. The resulting transgenic cattle lines showed rhLF expression levels between 0.4 and 2.5 g/L.…”

Section: Expression and Purification Of Rhlfmentioning

confidence: 99%

The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin

et al. 2005

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Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.4 A. To our knowledge, the first reported structure of a recombinant protein produced in milk of transgenic livestock. Even though rhLF contains oligomannose- and hybrid-type N-linked glycans next to complex-type glycans, which are the only glycans found on natural hLF, the structures are identical within the experimental error (r.m.s. deviation of only 0.28 A for the main-chain atoms). Of the differences in polymorphic amino acids between the natural and rhLF variant used, only the side-chain of Asp561 could be modeled into the rhLF electron density map. Taken together, the results confirm the structural integrity of the rhLF variant used in this study. It also confirms the validity of the transgenic cow mammary gland as a vehicle to produce recombinant human proteins.

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“…Lf is globular and consists of homologous N and C lobes (3,7,8), which can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) after digestion with trypsin (62). This experiment revealed binding of rIsdA to both lobes of Lf (Fig.…”

Section: Isda Is An Lf Binding Proteinmentioning

confidence: 99%

IsdA ProtectsStaphylococcus aureusagainst the Bactericidal Protease Activity of Apolactoferrin

Clarke

Foster

2008

Infect Immun

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An important facet of the Staphylococcus aureus host-pathogen interaction is the ability of the invading bacterium to evade host innate defenses, particularly the cocktail of host antimicrobial peptides. In this work, we showed that IsdA, a surface protein of S. aureus which is required for nasal colonization, binds to lactoferrin, the most abundant antistaphylococcal polypeptide in human nasal secretions. The presence of IsdA on the surface of S. aureus confers resistance to killing by lactoferrin. In addition, the bactericidal activity of lactoferrin was inhibited by addition of phenylmethylsulfonyl fluoride, implicating the serine protease activity of lactoferrin in the killing of S. aureus. Recombinant IsdA was a competitive inhibitor of lactoferrin protease activity. Reciprocally, antibody reactive to IsdA enhanced killing of S. aureus. Thus, IsdA can protect S. aureus against lactoferrin and acts as a protease inhibitor.

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The role of N‐linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis

Cited by 112 publications

References 24 publications

Lactoferrin Inhibits Porphyromonas gingivalis Proteinases and Has Sustained Biofilm Inhibitory Activity

Lactoferrin Inhibits Porphyromonas gingivalis Proteinases and Has Sustained Biofilm Inhibitory Activity

The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin

IsdA ProtectsStaphylococcus aureusagainst the Bactericidal Protease Activity of Apolactoferrin

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