Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

N-linked glycan truncation causes enhanced clearance of plasma-derived von Willebrand factor.

Essentials von Willebrands factor (VWF) glycosylation plays a key role in modulating in vivo clearance. VWF glycoforms were used to examine the role of specific glycan moieties in regulating clearance. Reduction in sialylation resulted in enhanced VWF clearance through asialoglycoprotein receptor. Progressive VWF N-linked glycan trimming resulted in increased macrophage-mediated clearance. Click to hear Dr Denis discuss clearance of von Willebrand factor in a free presentation from the ISTH Academy SUMMARY: Background Enhanced von Willebrand factor (VWF) clearance is important in the etiology of both type 1 and type 2 von Willebrand disease (VWD). In addition, previous studies have demonstrated that VWF glycans play a key role in regulating in vivo clearance. However, the molecular mechanisms underlying VWF clearance remain poorly understood. Objective To define the molecular mechanisms through which VWF N-linked glycan structures influence in vivo clearance. Methods By use of a series of exoglycosidases, different plasma-derived VWF (pd-VWF) glycoforms were generated. In vivo clearance of these glycoforms was then assessed in VWF-/- mice in the presence or absence of inhibitors of asialoglycoprotein receptor (ASGPR), or following clodronate-induced macrophage depletion. Results Reduced amounts of N-linked and O-linked sialylation resulted in enhanced pd-VWF clearance modulated via ASGPR. In addition to this role of terminal sialylation, we further observed that progressive N-linked glycan trimming also resulted in markedly enhanced VWF clearance. Furthermore, these additional N-linked glycan effects on clearance were ASGPR-independent, and instead involved enhanced macrophage clearance that was mediated, at least in part, through LDL receptor-related protein 1. Conclusion The carbohydrate determinants expressed on VWF regulate susceptibility to proteolysis by ADAMTS-13. In addition, our findings now further demonstrate that non-sialic acid carbohydrate determinants expressed on VWF also play an unexpectedly important role in modulating in vivo clearance through both hepatic ASGPR-dependent and macrophage-dependent pathways. In addition, these data further support the hypothesis that variation in VWF glycosylation may be important in the pathophysiology underlying type 1C VWD.

High Affinity Binding of the Receptor-associated Protein D1D2 Domains with the Low Density Lipoprotein Receptor-related Protein (LRP1) Involves Bivalent Complex Formation: CRITICAL ROLES OF LYSINES 60 AND 191.

The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that binds and mediates the endocytosis of numerous structurally diverse ligands. Currently, the basis for ligand recognition by LRP1 is not well understood. LRP1 requires a molecular chaperone, termed the receptor-associated protein (RAP), to escort the newly synthesized receptor from the endoplasmic reticulum to the Golgi. RAP is a three-domain protein that contains the following two high affinity binding sites for LRP1: one is located within domains 1 and 2, and one is located in its third domain. Studies on the interaction of the RAP third domain with LRP1 reveal critical contributions by lysine 256 and lysine 270 for this interaction. From these studies, a model for ligand recognition by this class of receptors has been proposed. Here, we employed surface plasmon resonance to investigate the binding of RAP D1D2 to LRP1. Our results reveal that the high affinity of D1D2 for LRP1 results from avidity effects mediated by the simultaneous interactions of lysine 60 in D1 and lysine 191 in D2 with sites on LRP1 to form a bivalent D1D2-LRP1 complex. When lysine 60 and 191 are both mutated to alanine, the binding of D1D2 to LRP1 is ablated. Our data also reveal that D1D2 is able to bind to a second distinct site on LRP1 to form a monovalent complex. The studies confirm the canonical model for ligand recognition by this class of receptors, which is initiated by pairs of lysine residues that dock into acidic pockets on the receptor.

Generation of a Potent Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Antagonist by Engineering a Stable Form of the Receptor-associated Protein (RAP) D3 Domain.

The low density lipoprotein receptor-related protein 1 (LRP1) is a member of the low density lipoprotein receptor family and plays important roles in a number of physiological and pathological processes. Expression of LRP1 requires the receptor-associated protein (RAP), a molecular chaperone that binds LRP1 and other low density lipoprotein receptor family members in the endoplasmic reticulum and traffics with them to the Golgi where the acidic environment causes its dissociation. Exogenously added RAP is a potent LRP1 antagonist and binds to LRP1 on the cell surface, preventing ligands from binding. Following endocytosis, RAP dissociates in the acidic endosome, allowing LRP1 to recycle back to the cell surface. The acid-induced dissociation of RAP is mediated by its D3 domain, a relatively unstable three-helical bundle that denatures at pH <6.2 due to protonation of key histidine residues on helices 2 and 3. To develop an LRP1 inhibitor that does not dissociate at low pH, we introduced a disulfide bond between the second and third helices in the RAP D3 domain. By combining this disulfide bond with elimination of key histidine residues, we generated a stable RAP molecule that is resistant to both pH- and heat-induced denaturation. This molecule bound to LRP1 with high affinity at both neutral and acidic pH and proved to be a potent inhibitor of LRP1 function both in vitro and in vivo, suggesting that our stable RAP molecule may be useful in multiple pathological settings where LRP1 blockade has been shown to be effective.

A novel chemical footprinting approach identifies critical lysine residues involved in the binding of receptor-associated protein to cluster II of LDL receptor-related protein.

Tandem mass tags (TMTs) were utilized in a novel chemical footprinting approach to identify lysine residues that mediate the interaction of receptor-associated protein (RAP) with cluster II of LDL (low-density lipoprotein) receptor (LDLR)-related protein (LRP). The isolated RAP D3 domain was modified with TMT-126 and the D3 domain-cluster II complex with TMT-127. Nano-LC-MS analysis revealed reduced modification with TMT-127 of peptides including Lys(256), Lys(270) and Lys(305)-Lys(306) suggesting that these residues contribute to cluster II binding. This agrees with previous findings that Lys(256) and Lys(270) are critical for binding cluster II sub-domains [Fisher, Beglova and Blacklow (2006) Mol. Cell 22, 277-283]. Cluster II-binding studies utilizing D3 domain variants K(256)A, K(305)A and K(306)A now showed that Lys(306) contributes to cluster II binding as well. For full-length RAP, we observed that peptides including Lys(60), Lys(191), Lys(256), Lys(270) and Lys(305)-Lys(306) exhibited reduced modification with TMT in the RAP-cluster II complex. Notably, Lys(60) has previously been implicated to mediate D1 domain interaction with cluster II. Our results suggest that also Lys(191) of the D2 domain contributes to cluster II binding. Binding studies employing the RAP variants K(191)A, K(256)A, K(305)A and K(306)A, however, revealed a modest reduction in cluster II binding for the K(256)A variant only. This suggests that the other lysine residues can compensate for the absence of a single lysine residue for effective complex assembly. Collectively, novel insight has been obtained into the contribution of lysine residues of RAP to cluster II binding. In addition, we propose that TMTs can be utilized to identify lysine residues critical for protein complex formation.

Quantitative dissection of the binding contributions of ligand lysines of the receptor-associated protein (RAP) to the low density lipoprotein receptor-related protein (LRP1).

Although lysines are known to be critical for ligand binding to LDL receptor family receptors, relatively small reductions in affinity have been found when such lysines have been mutated. To resolve this paradox, we have examined the specific binding contributions of four lysines, Lys-253, Lys-256, Lys-270, and Lys-289, in the third domain (D3) of receptor-associated protein (RAP), by eliminating all other lysine residues. Using D3 variants containing lysine subsets, we examined binding to the high affinity fragment CR56 from LRP1. With this simplification, we found that elimination of the lysine pairs Lys-253/Lys-256 and Lys-270/Lys-289 resulted in increases in Kd of 1240- and 100,000-fold, respectively. Each pair contributed additively to overall affinity, with 61% from Lys-270/Lys-289 and 39% from Lys-253/Lys-256. Furthermore, the Lys-270/Lys-289 pair alone could bind different single CR domains with similar affinity. Within the pairs, binding contributions of Lys-270 ≫ Lys-256 > Lys-253 ∼ Lys-289 were deduced. Importantly, however, Lys-289 could significantly compensate for the loss of Lys-270, thus explaining how previous studies have underestimated the importance of Lys-270. Calorimetry showed that favorable enthalpy, from Lys-256 and Lys-270, overwhelmingly drives binding, offset by unfavorable entropy. Our findings support a mode of ligand binding in which a proximal pair of lysines engages the negatively charged pocket of a CR domain, with two such pairs of interactions (requiring two CR domains), appropriately separated, being alone sufficient to provide the low nanomolar affinity found for most protein ligands of LDL receptor family members.

Mapping the binding region on the low density lipoprotein receptor for blood coagulation factor VIII.

Low density lipoprotein receptor (LDLR) was shown to mediate clearance of blood coagulation factor VIII (FVIII) from the circulation. To elucidate the mechanism of interaction of LDLR and FVIII, our objective was to identify the region of the receptor necessary for binding FVIII. Using surface plasmon resonance, we found that LDLR exodomain and its cluster of complement-type repeats (CRs) bind FVIII in the same mode. This indicated that the LDLR site for FVIII is located within the LDLR cluster. Similar results were obtained for another ligand of LDLR, α-2-macroglobulin receptor-associated protein (RAP), a common ligand of receptors from the LDLR family. We further generated a set of recombinant fragments of the LDLR cluster and assessed their structural integrity by binding to RAP and by circular dichroism. A number of fragments overlapping CR.2-5 of the cluster were positive for binding RAP and FVIII. The specificity of these interactions was tested by site-directed mutagenesis of conserved tryptophans within the LDLR fragments. For FVIII, the specificity was also tested using a single-chain variable antibody fragment directed against the FVIII light chain as a competitor. Both cases resulted in decreased binding, thus confirming its specificity. The mutagenic study also showed an importance of the conserved tryptophans in LDLR for both ligands, and the competitive binding results showed an involvement of the light chain of FVIII in its interaction with LDLR. In conclusion, the region of CR.2-5 of LDLR was defined as the binding site for FVIII and RAP.

Receptor-associated protein (RAP) has two high-affinity binding sites for the low-density lipoprotein receptor-related protein (LRP): consequences for the chaperone functions of RAP.

RAP (receptor-associated protein) is a three domain 38 kDa ER (endoplasmic reticulum)-resident protein that is a chaperone for the LRP (low-density lipoprotein receptor-related protein). Whereas RAP is known to compete for binding of all known LRP ligands, neither the location, the number of binding sites on LRP, nor the domains of RAP involved in binding is known with certainty. We have systematically examined the binding of each of the three RAP domains (D1, D2 and D3) to tandem and triple CRs (complement-like repeats) that span the principal ligand-binding region, cluster II, of LRP. We found that D3 binds with low nanomolar affinity to all (CR)2 species examined. Addition of a third CR domain increases the affinity for D3 slightly. A pH change from 7.4 to 5.5 gave only a 6-fold increase in Kd for D3 at 37 degrees C, whereas temperature change from 22 degrees C to 37 degrees C has a similar small effect on affinity, raising questions about the recently proposed D3-destabilization mechanism of RAP release from LRP. Surprisingly, and in contrast to literature suggestions, D1 and D2 also bind to most (CR)2 and (CR)3 constructs with nanomolar affinity. Although this suggested that there might be three high-affinity binding sites in RAP for LRP, studies with intact RAP showed that only two binding sites are available in the intact chaperone. These findings suggest a new model for RAP to function as a folding chaperone and also for the involvement of YWTD domains in RAP release from LRP in the Golgi.

Targeting of adenovirus vectors to the LRP receptor family with the high-affinity ligand RAP via combined genetic and chemical modification of the pIX capsomere.

Adenovirus (Ad) vector targeting requires presentation of specific ligands on the virion's surface. Geneti-chemical targeting is based on the genetic introduction of cysteine residues bearing reactive thiol groups into solvent-accessible capsomeres of the virion and subsequent chemical coupling of ligands. Here, we exploited this technology to modify the pIX capsomere with high-affinity ligands. Genetic introduction of C-terminal cysteines to pIX allowed for specific coupling of full-length proteins to the virion, while not affecting vector production. Direct comparison of the two high-affinity ligands receptor- associated protein (RAP) and transferrin (Tf) revealed that targeting after coupling of a high-affinity ligand to pIX presumably requires release of the ligand from its receptor after cell entry. In addition, data obtained by live cell imaging of labeled vector particles demonstrated that coupling of very large proteins to pIX can impair intracellular vector particle trafficking. Finally, we demonstrate that the geneti-chemical targeting technology is suitable for in vivo targeting to liver after intravenous injection. Our data provide significant insight into basic requirements for successful targeting of pIX-modified Ad vectors.

Unfolding of the RAP-D3 helical bundle facilitates dissociation of RAP-receptor complexes.

The receptor-associated protein (RAP) functions as an escort protein for receptors of the low-density lipoprotein receptor (LDLR) family by preventing premature intracellular binding of ligands and assisting with delivery of mature receptors to the cell surface. The modulation of affinity by pH is believed to play an important role in the escort function of RAP, because RAP binds tightly to proteins of the LDLR family at near-neutral pH early in the secretory pathway where its high affinity precludes premature binding of ligands but then dissociates from bound receptors at the lower pH of the Golgi compartment. The third domain of RAP (RAP-D3), which forms a three-helix bundle, is sufficient to reconstitute the escort activity. Here, we test the hypothesis that low-pH induced unfolding of the RAP-D3 helical bundle facilitates dissociation of RAP-receptor complexes. First, variants of RAP-D3 resistant to low pH-induced unfolding were constructed by replacing interior histidine residues with phenylalanines. In contrast to native RAP-D3, which exhibits an unfolding pKa of 6.3 and a Tm of 42 degrees C, the most hyperstable variant of RAP-D3, in which four histidine residues are replaced with phenylalanine, has an unfolding pKa of 4.8, and a Tm of 58 degrees C. The phenylalanine substitutions in RAP-D3 confer increased stability to pH-induced dissociation of complexes formed between RAP-D3 and a two-repeat fragment of the LDLR (LA3-4). When introduced into full-length RAP, the four mutations that confer hyperstability on RAP-D3 interfere with transport of endogenous LRP-1 to the cell surface in a dominant negative fashion under conditions where expression of normal RAP has no effect on LRP-1 transport. Our studies support a model in which low pH-dependent unfolding of RAP-D3 facilitates dissociation of RAP from the LA repeats of LDLR family proteins in the mildly acidic pH of the Golgi.

Functional mimicry of the LDL receptor-associated protein through multimerization of a minimized third domain.

The LDL receptor-associated protein (RAP) is a ligand for the LDL receptor-related protein (LRP1). The first and third domains of RAP can each bind to one of many sequence-related pairs of complement-type repeats (CR) found within the LRP1 ectodomain. Multiple sites of interaction between the multivalent RAP ligand and the multivalent LRP1 receptor yield strong binding avidity for the complex. The third domain of RAP can be significantly truncated, with material retention of monovalent CR pair-binding affinity, provided that the minimized sequence is stabilized with an intramolecular disulfide bond. We demonstrate that the avidity of full-length RAP for LRP1 in vitro can be partially reconstituted by assembly of truncated, disulfide-linked RAP peptides on tetravalent streptavidin or bivalent immunoglobulin scaffolds. The peptide complex with streptavidin shows pronounced hepatotropism in vivo, replicating the biodistribution of full-length RAP.

Versatility in ligand recognition by LDL receptor family proteins: advances and frontiers.

Proteins of the low-density lipoprotein receptor family transport cholesterol-carrying particles into cells, clear protease-inhibitor complexes from the circulation, participate in biological signaling cascades, and even serve as viral receptors. These receptors utilize clusters of cysteine-rich LDL receptor type-A (LA) modules to bind many of their ligands. Recent structures show that these modules typically exhibit a characteristic binding mode to recognize their partners, relying primarily on electrostatic complementarity and avidity effects. The dominant contribution of electrostatic interactions with small interface areas in these complexes allows binding to be regulated by changes in pH via at least two distinct mechanisms. The structure of the subtilisin/kexin family protease PCSK9, a newly identified molecular partner of the LDLR also implicated in LDL-cholesterol homeostasis, also raises the possibility that the LDLR and its related family members may employ other strategies for pH-sensitive binding that have yet to be uncovered.

Minimization of the third domain of the LDL receptor-associated protein (RAP).

The third domain of the low-density lipoprotein receptor-associated protein (RAP d3) binds with high-affinity to pairs of complement-type repeats (CR) within the LDLR family of receptors. Structural analyses have defined the contact surface between RAP d3 and a CR pair from the low-density lipoprotein receptor (LDLR). Much of the sequence of RAP d3 has been proposed to stabilize the receptor-binding region without participating directly in formation of the contact surface. We have developed a truncated version of RAP d3 in which these scaffolding regions are excised and replaced with a single, intramolecular disulfide bond. This substitution allows for deletion of as much as a third of the RAP d3 sequence with substantial retention of receptor-binding ability.

The structure of receptor-associated protein (RAP).

The receptor-associated protein (RAP) is a molecular chaperone that binds tightly to certain newly synthesized LDL receptor family members in the endoplasmic reticulum (ER) and facilitates their delivery to the Golgi. We have adopted a divide-and-conquer strategy to solve the structures of the individual domains of RAP using NMR spectroscopy. We present here the newly determined structure of domain 2. Based on this structure and the structures of domains 1 and 3, which were solved previously, we utilized experimental small-angle neutron scattering (SANS) data and a novel simulated annealing protocol to characterize the overall structure of RAP. The results reveal that RAP adopts a unique structural architecture consisting of three independent three-helix bundles that are connected by long and flexible linkers. The flexible linkers and the quasi-repetitive structural architecture may allow RAP to adopt various possible conformations when interacting with the LDL receptors, which are also made of repetitive substructure units.

Optimal expression condition of recombinant RAP.

In order to construct the expression recombinant of human receptor associated protein (RAP), optimize its expression condition and obtain the recombinant protein after expression with high efficiency, two prokaryotic expression vectors-pT7-PL and pET-28a(+) were used to construct the expression recombinant containing RAP cDNA, and the expression efficiency of two kinds of expression E. coli of BL21 strains was compared. The effect of different induction conditions on the expression of recombinant RAP was observed. After recombinant protein was purified with Ni(+) -nitrilotriacetic acid (Ni(+) -NTA) affinity chromatogram, its binding ability with microphage was observed. The results showed that two recombinant plasmids both obtained high expression of RAP. The expression levels of RAP in plasmid pT7-PL-RAP in BL21 (DE3, plysS) strain were significantly higher than in BL21 (DE3) strain. The expression of pT7-PL-RAP in the presence of chloramphenicol was higher than in the absence of chloramphenicol, and most of the inducible expressed RAP was soluble. The RAP which was purified by Ni(+) -NTA resin could strongly bind with the RAW264.7 cells rich in low density lipoprotein receptor (LDLR) family receptors. It was concluded that the expression condition of recombinant RAP was optimized and functional RAP was obtained, which offered a good foundation for the further production of RAP as research tool.

The switch on the RAPper's necklace...

The biosynthesis and export of LDL receptor-related proteins rely on specialized chaperones in the endoplasmic reticulum. Two recent papers in Molecular Cell by Fisher et al. (2006) and Lee et al. (2006) reveal a novel mechanism by which one of these chaperones, the receptor-associated protein RAP, accomplishes this task.

Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors.

Proteins of the low-density lipoprotein receptor (LDLR) family are remarkable in their ability to bind an extremely diverse range of protein and lipoprotein ligands, yet the basis for ligand recognition is poorly understood. Here, we report the 1.26 A X-ray structure of a complex between a two-module region of the ligand binding domain of the LDLR and the third domain of RAP, an escort protein for LDLR family members. The RAP domain forms a three-helix bundle with two docking sites, one for each LDLR module. The mode of recognition at each site is virtually identical: three conserved, calcium-coordinating acidic residues from each LDLR module encircle a lysine side chain protruding from the second helix of RAP. This metal-dependent mode of electrostatic recognition, together with avidity effects resulting from the use of multiple sites, represents a general binding strategy likely to apply in the binding of other basic ligands to LDLR family proteins.

Interaction of lipoprotein lipase and receptor-associated protein.

Receptor-associated protein (RAP) is a recognized chaperone/escort protein for members of the low density lipoprotein receptor family. In this report, we show that RAP binds to lipoprotein lipase (LPL) and may play a role in the maturation of LPL. Binding of highly purified RAP to LPL was demonstrated in vitro by solid phase assays, surface plasmon resonance, and rate zonal centrifugation. The dissociation constant for this interaction measured by the first two techniques ranged between 2.4 and 13 nM, values similar to those reported for the binding of RAP to LRP or gp330. The specificity of the interaction was demonstrated by competition with a panel of LPL monoclonal antibodies. Rate zonal centrifugation demonstrated the presence of a stable complex with an apparent Mr consistent with the formation of a complex between monomeric LPL and RAP. RAP x LPL complexes were co-immunoprecipitated in adipocyte lysates or from solutions of purified LPL and RAP. The interaction was also demonstrated in whole cells by cross-linking experiments. RAP-deficient adipocytes secreted LPL with a specific activity 2.5-fold lower than the lipase secreted by control cells. Heparin addition to cultured RAP-deficient adipocytes failed to stimulate LPL secretion in the medium, suggesting defective binding of the lipase to the plasma membrane. These studies demonstrate that RAP binds to LPL with high affinity both in purified systems and cell extracts and that RAP-deficient adipocytes secrete poorly assembled LPL. A function of RAP may be to prevent premature interaction of LPL with binding partners in the secretory pathway, namely LRP and heparan sulfate proteoglycan.