The limited capacity of current bioreactors has led the biopharmaceutical industry to investigate alternative protein expression systems. The milk of transgenic cattle may provide an attractive vehicle for large-scale production of biopharmaceuticals, but there have been no reports on the characteristics of such recombinant proteins. Here we describe the production of recombinant human lactoferrin (rhLF), an iron-binding glycoprotein involved in innate host defense, at gram per liter concentrations in bovine milk. Natural hLF from human milk and rhLF had identical iron-binding and -release properties. Although natural hLF and rhLF underwent differential N-linked glycosylation, they were equally effective in three different in vivo infection models employing immunocompetent and leukocytopenic mice, and showed similar localization at sites of infection. Taken together, the results illustrate the potential of transgenic cattle in the large-scale production of biopharmaceuticals.

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.

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.

Lactoferrin (LF), a cationic 80-kDa protein present in polymorphonuclear leukocytes and in mucosal secretions, is known to have antibacterial effects on gram-negative bacteria, with a concomitant release of lipopolysaccharides (LPS, endotoxin). In addition, LF is known to decrease LPS-induced cytokine release by monocytes and LPS priming of polymorphonuclear leukocytes. Its mechanism of action is incompletely understood. We have now demonstrated by in vitro-binding studies that LF binds directly to isolated lipid A and intact LPS of clinically relevant serotypes of the species which most frequently cause bacteremia (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), as well as to lipid A and LPS of mucosal pathogens (among others, Neisseria meningitides and Haemophilus influenzae). Binding to LPS was inhibitable by lipid A and polymyxin B but not by KDO (3-deoxy-D-manno-octulosonate), a glycoside residue present in the inner core of LPS. Binding of LF to lipid A was saturable, and an affinity constant of 2 x 109 M-1 was calculated for the LF-lipid A interaction. Our data may explain, in part, the mechanism whereby LF exerts its antibacterial and anti-endotoxic effects. Further studies on the ability of LF to block the detrimental effects of LPS, both in vitro and in vivo, are warranted.

Lactoferrin (LF) is an iron-binding glycoprotein of the innate host defence system. To elucidate the role of N-linked glycosylation in protection of LF against proteolysis, we compared the tryptic susceptibility of human LF (hLF) variants from human milk, expressed in human 293(S) cells or in the milk of transgenic mice and cows. The analysis revealed that recombinant hLF (rhLF) with mutations Ile130-->Thr and Gly404-->Cys was about twofold more susceptible than glycosylated and unglycosylated variants with the naturally occurring Ile130 and Gly404. Hence, N-linked glycosylation is not involved in protection of hLF against tryptic proteolysis. Apparently, the previously reported protection by N-linked glycosylation of hLF [van Berkel, P.H.C., Geerts, M.E.J., van Veen, H.A., Kooiman, P.M., Pieper, F., de Boer, H.A. & Nuijens, J.H. (1995) Biochem. J. 312, 107-114] is restricted to rhLF containing the Thr130 and Cys404. Comparison of the tryptic proteolysis of hLF and bovine LF (bLF) revealed that hLF is about 100-fold more resistant than bLF. Glycosylation variants A and B of bLF differed by about 10-fold in susceptibility to trypsin. This difference is due to glycosylation at Asn281 in bLF-A. Hence, glycosylation at Asn281 protects bLF against cleavage by trypsin at Lys282.

Human lactoferrin (hLF) is a glycoprotein involved in the host defence against infection and excessive inflammation. Our objective was to determine to what extent each of the three sequons for N-linked glycosylation in hLF is actually used. Human kidney-derived 293(S) cell lines expressing recombinant hLF (rhLF) or glycosylation-site mutants were produced. The mutations involved replacement of asparagine residues with glutamine at one or more sequons for N-glycosylation (Asn138, Asn479 and Asn624). Comparative SDS/PAGE analyses of rhLF, mutated rhLF and human-milk-derived (natural) hLF led us to propose that glycosylation of hLF occurs at two sites (at Asn138 and Asn479) in approx. 85% of all hLF molecules. Glycosylation at a single site (Asn479) or at all three sites occurs in approx, 5% and 9% of hLF respectively. The extent of glycosylation at Asn624 was increased to approx. 29% and 40% of Asn479 and Asn138/479 mutant molecules respectively, which indicates that glycosylation at Asn624 in natural hLF might be limited by glycosylation at Asn479. The presence in supernatant of unglycosylated hLF (approx. 60% of the total) after mutations of Asn138 and Asn479 suggests that glycosylation of hLF is not an absolute requirement for its secretion. The pronounced degradation of unglycosylated hLF in supernatant after mutation at all three glycosylation sites (Asn138/479/624 mutant) but not after mutation at both Asn138 and Asn479 suggests that an altered conformation rather than the lack of glycosylation has rendered the Asn138/479/624 mutant susceptible to intra- and/or extra-cellular degradation.

Human lactoferrin (hLF) is an iron-binding protein involved in host defense against infection and severe inflammation. Transgenic mice were produced harboring either hLF cDNA or genomic hLF sequences fused to regulatory elements of the bovine ␣S 1 casein gene. Recombinant hLF expressed in the milk of transgenic mice (transgenic hLF) was compared with natural (human milk-derived) hLF. Immunological identity of the two forms was shown by double antibody immunoassays and the absence of an anti-hLF antibody response in transgenic mice on hyperimmunization with natural hLF. Mono S cation-exchange chromatography and N-terminal protein sequencing of transgenic and natural hLF revealed identical cationicity and N-terminal sequences. SDS-polyacrylamide gel electrophoresis and absorbance measurements of purified transgenic hLF showed this protein was 90% saturated with iron, whereas natural hLF is only 3% saturated. The pH-mediated release of iron from transgenic hLF was not different from that of iron-saturated natural hLF. Unsaturated transgenic hLF could be completely resaturated upon addition of iron. Slight differences in mobility between transgenic and natural hLF on SDS-polyacrylamide gel electrophoresis were abolished by enzymatic deglycosylation. Binding of transgenic and natural hLF to a range of ligands, including bacterial lipopolysaccharide, heparin, single-stranded DNA, Cibacron blue FG 3A, and lectins, was not different.Based on these observations, we anticipate that (unsaturated) rhLF and natural hLF will exert similar, if not identical, antibacterial and anti-inflammatory activity in vivo.

T lymphocytes can be induced to produce interleukin (IL)-2 and proliferate upon T cell receptor (TcR) occupancy together with a CD28-induced co-stimulatory signal. The T cell surface molecule CD28 is believed to function as a regulator in T cell activation at both the level of lymphokine mRNA stabilization and gene transcription. Activation of IL-2 gene transcription via CD28 has been shown to be mediated through a kappa B-like sequence, called CD28RE. DNA binding analysis revealed that the CD28-induced signal is involved in the induction of CD28RE binding activity. Here, we demonstrate that the induction of CD28RE binding activity is not specific for the CD28-induced signal. Our data indicate that distinct mitogenic T cell activation signals converge on the induction of CD28RE binding activity, and suggest a crucial role for this activity in the IL-2 enhancer responsiveness to different modes of T cell activation.