| Human lactoferrin (h-LF) is widely reported to be involved in numerous biological functions like iron transport, anti-microbial defense, anti-tumor mechanisms and immune system regulation. Currently there is an extensive interest in recombinant sources of this glycoprotein for pharmaceutical and neutraceutical purposes. Recombinant human lactoferrin from Aspergillus awamori (Rh-LF) is a major scalable source presently undergoing several clinical trials. In this study, we comprehensively characterized the structure and also studied the functions of N-glycans in Rh-LF.;Lys-C peptide map LC-MS analysis of Rh-LF localized N-linked oligosaccharides to Asn138, Asn479 and Asn624. The third site at Asn624 was partially occupied with high-mannose oligosaccharides unlike native human lactoferrin, wherein Asn624 is typically vacant. N -glycan profiling using NP-HPLC coupled with ESI-TOF mass spectrometry analysis revealed that high mannose glycans of Rh-LF were composed of 6 - 14 mannose moieties. SPR assays showed that presence or absence of glycans had no effect on binding affinity towards Intelectin , a major in vivo lactoferrin receptor and the binding affinity of deglycosyalted Rh-LF was observed to be close to that of Rh-LF and native human Lactoferrin. SEC analysis revealed deglycosylation did not resulted in increased aggregation of Rh-LF, signifying lack of role of glycans in Rh-LF stability post secretion. |
