Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells

Abstract

Lactoferrin (Lf), a multifunctional molecule present in mammalian secretions and blood, plays important roles in host defense and cancer. Indeed, Lf has been reported to inhibit the proliferation of cancerous mammary gland epithelial cells and manifest a potent antiviral activity against human immunodeficiency virus and human cytomegalovirus. The Lf‐binding sites on the cell surface appear to be proteoglycans and other as yet undefined protein(s). Here, we isolated a Lf‐binding 105 kDa molecular mass protein from cell extracts and identified it as human nucleolin. Medium–affinity interactions (≈ 240 nm) between Lf and purified nucleolin were further illustrated by surface plasmon resonance assays. The interaction of Lf with the cell surface‐expressed nucleolin was then demonstrated through competitive binding studies between Lf and the anti‐human immunodeficiency virus pseudopeptide, HB‐19, which binds specifically surface‐expressed nucleolin independently of proteoglycans. Interestingly, binding competition studies between HB‐19 and various Lf derivatives in proteoglycan‐deficient hamster cells suggested that the nucleolin‐binding site is located in both the N‐ and C‐terminal lobes of Lf, whereas the basic N‐terminal region is dispensable. On intact cells, Lf co‐localizes with surface nucleolin and together they become internalized through vesicles of the recycling/degradation pathway by an active process. Morever, a small proportion of Lf appears to translocate in the nucleus of cells. Finally, the observations that endocytosis of Lf is inhibited by the HB‐19 pseudopeptide, and the lack of Lf endocytosis in proteoglycan‐deficient cells despite Lf binding, point out that both nucleolin and proteoglycans are implicated in the mechanism of Lf endocytosis.

Midkine, a cytokine that inhibits HIV infection by binding to the cell surface expressed nucleolin

“…The fact that HIV attachment is inhibited by two agents which do not compete, illustrates that virus attachment should be coordinated on one hand by heparan sulfate proteoglycans, and on the other hand by nucleolin. Consistent with the critical role of surface nucleolin in the initial steps of HIV infection, the HB-19 pseudopeptide and several physiological ligands of nucleolin (Midkine, Pleiotrophin, lactoferrin) inhibit HIV infection by blocking HIV particle attachment to CD4+ target cells [2][3][4][5][6].…”

“…In contrast to the high affinity receptors, the cell-surface expressed nucleolin is the only known low affinity receptor of MK [4]. Besides MK, surface nucleolin serves as a low affinity receptor for PTN [5] and lactoferrin [6].…”

Modulation of HIV Binding to Epithelial Cells and HIV Transfer from Immature Dendritic Cells to CD4 T Lymphocytes by Human Lactoferrin and its Major Exposed LF-33 Peptide

“…However, although both CD4 and coreceptors are essential for HIV-1 entry process, the initial association of HIV-1 particles to cells could occur in the absence of these receptors. Several observations pointed out that the attachment of HIV-1 particles to the cell surface occurs through interactions with heparan-sulfate proteoglycans and with the cell surface-expressed nucleolin [14][15][16], suggesting their possible involvement in HIV-1 attachment inhibition on epithelial cells by hLF. Thus, LF likely could exert its effect at the level of viral adsorption or penetration, in accordance with its highest effectiveness when administered before or simultaneous with the viral inoculum, as it occurs in vivo during breastfeeding and sexual transmission of HIV-1 [6].…”

Interaction of Human Lactoferrin with Cell Adhesion Molecules through RGD Motif Elucidated by Lactoferrin-binding Epitopes

“…Furthermore, the same group identified the associated hLF receptor gene (10). This receptor bound to hLF with a K d of 1 M. A nucleolar protein called nucleolin is also a potential hLF receptor (11). In this case, both the N-and C-lobes of LF contributed to the receptor binding, and the K d was 240 nM.…”