Characterization of Glycans in a Lactoferrin Isoform, Lactoferrin-a

Wei, Zheng; Nishimura, Toshihide; Yoshida, Shigeru

doi:10.3168/jds.s0022-0302(01)74712-1

Cited by 23 publications

(17 citation statements)

References 13 publications

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“…Bovine LF contains five N-linked glycosylation sites (Asn-233, -281, -368, -476, and -545) (55), and the majority of glycans are located in the N-terminal region of LF (33-kDa fragment); conse- . Each data point represents the mean and standard deviation of six biological replicates.…”

Section: Discussionmentioning

confidence: 99%

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: 99%

Lactoferrin Inhibits Porphyromonas gingivalis Proteinases and Has Sustained Biofilm Inhibitory Activity

Dashper

Pan

Veith

et al. 2012

Antimicrob Agents Chemother

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“…Although members of the LRP family are generally considered as endocytic receptors, LRP1 can also function as a signaling receptor [29]. The key to understanding the molecular basis of LF various activities is thought to reside in part according to patterns of glycosylation [37]. There are three possible N-linked glycosylation sites in hLF, one at Asn138, a second site at Asn479, and a third site at Asn624; differential utilization of these sites results in distinct glycosylation variants [30,35].…”

Section: Introductionmentioning

confidence: 99%

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

et al. 2008

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Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.

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“…AGP, which on the array was bound with high affinity by three FBA proteins, contains a heterogeneous mixture of five complex glycans in multiple isoforms (37). Lactoferrin consists of two isoforms, each containing two high mannose glycans with variable additional complex glycans (38). To model sulfated glycans we chose heparin, a disaccharide repeat of GlcNAc and glucuronic acid (GlcUA) sulfated at the C-6 position and N position of GlcNAc, and at the C-2 position of IdoUA, the epimerized form of GlcA (39).…”

Section: Divergent Binding To Model Glycans and Glycoproteins-mentioning

confidence: 99%

Diversity in Tissue Expression, Substrate Binding, and SCF Complex Formation for a Lectin Family of Ubiquitin Ligases

Glenn

Nelson

Wen³

et al. 2008

Journal of Biological Chemistry

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Post-translational modification of proteins regulates many cellular processes. Some modifications, including N-linked glycosylation, serve multiple functions. For example, the attachment of N-linked glycans to nascent proteins in the endoplasmic reticulum facilitates proper folding, whereas retention of high mannose glycans on misfolded glycoproteins serves as a signal for retrotranslocation and ubiquitin-mediated proteasomal degradation. Here we examine the substrate specificity of the only family of ubiquitin ligase subunits thought to target glycoproteins through their attached glycans. The five proteins comprising this FBA family (FBXO2, FBXO6, FBXO17, FBXO27, and FBXO44) contain a conserved G domain that mediates substrate binding. Using a variety of complementary approaches, including glycan arrays, we show that each family member has differing specificity for glycosylated substrates. Collectively, the F-box proteins in the FBA family bind high mannose and sulfated glycoproteins, with one FBA protein, FBX044, failing to bind any glycans on the tested arrays. Site-directed mutagenesis of two aromatic amino acids in the G domain demonstrated that the hydrophobic pocket created by these amino acids is necessary for high affinity glycan binding. All FBA proteins co-precipitated components of the canonical SCF complex (Skp1, Cullin1, and Rbx1), yet FBXO2 bound very little Cullin1, suggesting that FBXO2 may exist primarily as a heterodimer with Skp1. Using subunit-specific antibodies, we further demonstrate marked divergence in tissue distribution and developmental expression. These differences in substrate recognition, SCF complex formation, and tissue distribution suggest that FBA proteins play diverse roles in glycoprotein quality control.

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Characterization of Glycans in a Lactoferrin Isoform, Lactoferrin-a

Cited by 23 publications

References 13 publications

Lactoferrin Inhibits Porphyromonas gingivalis Proteinases and Has Sustained Biofilm Inhibitory Activity

Lactoferrin Inhibits Porphyromonas gingivalis Proteinases and Has Sustained Biofilm Inhibitory Activity

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

Diversity in Tissue Expression, Substrate Binding, and SCF Complex Formation for a Lectin Family of Ubiquitin Ligases

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