Significance of heparin binding to basic residues in homologous to the amino terminus of hepatoma-derived growth factor and related proteins

Abstract

Hepatoma-derived growth factor (HDGF) recognizes cell surface heparan sulfate to promote its internalization though binding to its N-terminal HATH (homologous to amino terminus of HDGF) domain. HDGF-related proteins (HRPs) all have the HATH domain in their N terminus. In this study, we report on the commonality of heparin binding in all HRPs with a broad range of heparin-binding affinity: HRP-4 is the strongest binder, and the lens epithelium-derived growth factor shows a relatively weak binding, with binding affinities (K(D)) showing 30-fold difference in magnitude. With the HDGF HATH domain used as a model, residue K19 was the most critical basic residue in molecular recognition and protein internalization, and with its proximal proline-tryptophan-tryptophan-proline motif, coordinated a conformational change when binding to the heparin fragment. Other basic residues, K21, K61, K70, K72 and R79, confer added contribution in binding that the total ionic interaction from these residues represents more than 70% of the binding energy. Because the positive-charged residues are conserved in all HRP HATH domains, heparin binding outside of cells might be of equal importance for all HRPs in mediating downstream signaling; however, distinct effects and/or distribution might be associated with the varying affinities to heparin.

Thrombophilia Associated with Anti-DFS70 Autoantibodies

“…LEDGF has been described as a secreted protein with growth-factor properties, also responsible for resistance to thermal and oxidative stresses [ 30 ]. The biological effects of LEDGF have been shown to be increased in the presence of heparin [ 31 , 32 ]. The capacity of heparin to interact with LEDGF may indicate that the LEDGF/anti-DFS70 antibodies immune complex may play a role in the induction of thrombosis.…”

Heparin-Promoted Cellular Uptake of the Cell-Penetrating Glycosaminoglycan Binding Peptide, GBPECP, Depends on a Single Tryptophan

“…The heparin-derived fragment corresponding to the octasaccharide was used in this study. To prepare heparin octasaccharide, low-molecular-weight porcine intestinal heparin (Sigma-Aldrich Corporation) was digested with Flavobacterium heparinase I (EC 4.2.2.7; Sigma H-2519), and the depolymerized heparin oligosaccharides were separated on a gel filtration Bio-Gel P-10 column (Bio-Rad Laboratories) . The prepared heparin octasaccharide contained structural inhomogeneity that more than 10 types of sequences were detected in the preparation.…”

The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein–Protein Interaction

“…The domain consists of 100–150 amino acids and is found in a number of eukaryotic proteins that are involved in cell division, growth, and differentiation . The PWWP domain was first identified as a structural motif in the Wolf–Hirschhorn syndrome critical region proteins (WHSC1) , and further studied in hepatoma-derived growth factor (HDGF). − On the basis of sequence homology, the PWWP domains have been categorized into several classes, and more than 10 structures of the PWWP domains have been determined in recent years since the first structure was determined for the DNA methyltransferase DNMT3B . These PWWP domains consist of a five-stranded antiparallel β-barrel and an α-helix bundle in their C-termini, and the PWWP motif is located in the N-terminal part of β2 .…”

“…The PWWP domain has been reported to play distinct roles on both sides of the cell membrane. − However, the exact biological function of the PWWP domain remains controversial and has not yet been established. Three interacting partners have been commonly addressed, namely, methylated histone, DNA, and heparin/heparan sulfate. ,,, Methylated histone peptide has been reported to weakly bind a highly conserved hydrophobic cage on the PWWP domain surface, constituted by three aromatic residues: the Tyr/Phe residue before the PWWP motif, the third residue (Trp) of the PWWP motif, and another aromatic residue (Phe/Trp/His) in β3. , Many PWWP domains have DNA binding ability, allowing some proteins to behave as transcription factors in regulating a variety of developmental processes. − PWWP domains contain a characteristic electrostatic surface where conserved positively charged residues are clustered in a polarized patch next to the hydrophobic cage . The charged patch is responsible for binding both DNA and heparin. , Thus, the combination of the two binding sites provides the possible mechanism of chromatin binding in which the conserved hydrophobic cage anchors the methylated histone molecule, and the charged patch makes contact with the nucleic acid. ,− However, the PWWP DNA binding is nonspecific .…”

“…HRPs play dual roles on both sides of the plasma membrane by either binding to DNA in the nucleus as a transcriptional factor or interacting with membrane surface carbohydrates as a growth factor. ,− They were classified on the basis of the conserved N-terminal PWWP domain, usually called the HATH (homologous to the amino terminus of HDGF) domain. , Recent studies have confirmed the consensus role of HATHs in binding heparin, DNA, and methylated peptides, − ,,, whereas the functions of HRP C-terminal domains with different lengths and charges remain unknown . Different cellular effects have been reported for different HRPs.…”