Hexa-thiocarbamoyl Phenyl PEG5K Hb: Vasoactivity and Structure

A new hexaPEGylated hemoglobin, (TCP-PEG5K)₆-Hb (TCP, thiocarbamoyl phenyl) has been developed using PEG-phenyl-isothiocyanate and its vasoactivity and structure has been investigated. Of the six PEG5K chains of (TCP-PEG5K)₆-Hb, 4 are conjugated to the α-amino groups of Hb, and the other 2 chains are distributed on ε-amino groups, identified as Lys-40(α) (~45%), Lys-56(α) (~25%), and Lys-8(β) (~24%). The studies with hamster infused with a bolus of a 4 gm % solution of (TCP-PEG5K)₆-Hb equivalent to 10% of their blood volume have established that this new hexaPEGylated Hb is vasoinactive. The viscosity and the colloidal osmotic pressure of (TCP-PEG5K)₆-Hb at 4% is 1.9 cP and 69.7 mmHg, respectively. The molecular radius of (TCP-PEG5K)₆-Hb is about 4.6 nm and is significantly smaller than hexaPEGylated Hbs developed using other direct and extension arm facilitated PEGylation platform. The presence of an outside the central cavity intramolecular crosslink, succinimidophenyl-PEG2K between Cys-93(β, β′) in (TCP-PEG5K)₆-ββ-Hb strongly impacts its solution properties. These patterns of influence suggest that the inter-dimeric interactions in (TCP-PEG5K)₆-Hb is weakened just as with other direct PEGylation platforms, and (SP-PEG5K)₆-Hb generated by EAF-PEGylation is unique in not inducing this effect. A comparison of the properties of hexaPEGylated Hbs establishes that rigidity of the conjugation linkage between PEG and Hb plays a significant influence on the resultant dictating solution properties and/structure/conformation of PEG-Hb adduct.     

Non-hypertensive tetraPEGylated canine haemoglobin: correlation between PEGylation, O2 affinity and tissue oxygenation

TetraPEGylated canine Hb, [SP (succinimidophenyl)-PEG5K]4-canine-Hb, with PEGylation at its four reactive cysteine residues (a111 and b93) has been prepared and characterized. The hydrodynamic volume and the molecular radius of (SP-PEG5K)4-canine-Hb are intermediate to those of di- and hexaPEGylated human Hb as expected. However, the COP (colloidal osmotic pressure) of tetraPEGylated canine Hb is closer to that of hexaPEGylated human Hb than to that of diPEGylated human Hb. The O2 affinity of tetraPEGylated canine Hb is higher than that of canine Hb and comparable with that of hexaPEGylated Hb. The O2 affinity of tetraPEGylated canine Hb is not responsive to the presence of DPG (diphosphoglycerate) or chloride, but it retains almost full response to L-35, an allosteric effector that interacts at the aa-end of the central cavity. The tetraPEGylated canine Hb is vasoinactive in hamster in 10% top load infusion studies. It is also essentially non-hypertensive in an extreme exchange haemodilution protocol in hamster just as di- and hexaPEGylated human Hb. The O2 delivery by tetraPEGylated canine Hb is comparable with that of hexaPEGylated Hb but not as efficient as diPEGylated Hb. These results demonstrate that PEGylation-induced solution properties of PEG [poly(ethylene glycol)]-Hb conjugates are dictated by the level and chemistry of PEGylation and the interplay of these plays a critical role in tissue oxygenation. The studies imply the need to establish the right level (and/or pattern) of PEGylation and O2 affinity of Hb-PEG adducts in designing O2-carrying plasma volume expanders, and this remains the primary challenge in the design of PEGylated Hb as blood substitutes.     

Conjugation of Multiple Copies of Polyethylene Glycol to Hemoglobin Facilitated Through Thiolation: Influence on Hemoglobin Structure and Function

PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)₆-Hb, that carries ~six PEG5K chains/Hb – HexaPEGylated Hb. PEGylation increased the O₂ affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)₆-Hb, its molecular volume, O₂ affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)₂-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)₆-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)₂-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.     

Non-conservative surface decoration of hemoglobin: influence of neutralization of positive charges at PEGylation sites on molecular and functional properties of PEGylated hemoglobin

High hydrodynamic volume, high viscosity and high colloidal osmotic pressure (COP) of PEGylated hemoglobin (Hb) have been suggested to neutralize the vasoactivity of acellular Hb. Consequences of non-conservative PEGylation (positive charge of the amino groups at the PEGylation sites is neutralized) using succinimidyl-ester of propionic acid PEG5K on the properties of PEGylated Hb have now been investigated. Non-conservative PEGylation of Hb leads to a much higher increase in the COP and viscosity of Hb than conservative extension arm facilitated (EAF) PEGylation of Hb. Introduction of alphaalpha-fumaryl crosslinking decreased the COP of non-conservative PEGylated Hb by stabilization of interdimeric interactions. Compared to the EAF-PEGylated alphaalpha-fumaryl Hb, non-conservative PEGylated product shows a comparable COP and higher viscosity. Conservative PEGylation of alphaalpha-fumaryl Hb by reductive alkylation chemistry does not increase the COP to this level, but enhanced the molecular volume and viscosity comparable to EAF-PEGylated product. Thus, the molecular properties of PEGylated Hb can be fine tuned using different PEGylation platforms and provide a unique opportunity for the design of second generation PEGylated Hbs.     

Extension Arm Facilitated PEGylation of Hemoglobin: Correlation of the Properties with the Extent of PEGylation

Human hemoglobin (Hb) conjugated with six copies of PEG-5K is nonhypertensive. The hexaPEGylated Hb exhibits molecular size homogeneity in spite of the chemical heterogeneity with respect to the sites of conjugation (Manjula et al., 2005). In the present study, Hb conjugated with an average of 4, 6, 8 and 10 copies of PEG-5K chains have been generated using the extension arm facilitated PEGylation protocol. Except for the tetraPEGylated Hb, all the other products exhibit molecular size homogeneity. The molecular, colligative and functional properties of PEG-Hb conjugates have been correlated with the extent of PEGylation. The results imply that six copies of PEG-5K chains are accommodated on Hb without significant crowding on the molecular surface. As more copies of PEG-5K chains are conjugated to form octa and deca PEGylated Hb, the PEG-chains conjugated appear to undergo transition from a mushroom (compact) to a brush-like conformation (extended conformation) with a concomitant decrease in the propensity of the molecule to transition from oxy to deoxy conformation in the presence of allosteric effectors. The viscosity and the colloidal osmotic pressure of Hb increase with the number of the PEG-chains conjugated in an exponential fashion. The composition of the PEGylated Hb generated appears to be controlled by (i) high reactivity of thiol groups of the extension arms on Hb with maleimide-PEG, (ii) increase in the viscosity of the reaction mixture as the level of PEGylation increases and (iii) increased resistance induced by the PEG-shell of PEGylated Hb to accommodate more PEG-chains as the level of PEGylation increases. Potential implications of extent of PEGylation on the oxygen delivery by PEG-Hb conjugate in vivo have been discussed.     

Reversible protection of Cys-93(β) by PEG alters the structural and functional properties of the PEGylated hemoglobin

As a potential hemoglobin (Hb)-based oxygen carrier (HBOC), the PEGylated Hb has received much attention for its non-nephrotoxicity. However, PEGylation can adversely alter the structural and functional properties of Hb. The site of PEGylation is an important factor to determine the structure and function of the PEGylated Hb. Thus, protection of some sensitive residues of Hb from PEGylation is of great significance to develop the PEGylated Hb as HBOC. Here, Cys-93(β) of Hb was conjugated with 20 kDa polyethylene glycol (PEG20K) through hydrazone and disulfide bonds. Then, the conjugate was modified with PEG5K succinimidyl carbonate (PEG5K-SC) using acylation chemistry, followed by removal of PEG20K Hb with hydrazone hydrolysis and disulfide reduction. Reversible conjugation of PEG20K at Cys-93(β) can protect Lys-95(β), Val-1(α) and Lys-16(α) of Hb from PEGylation with PEG5K-SC. The autoxidation rate, oxygen affinity, structural perturbation and tetramer instability of the PEGylated Hb were significantly decreased upon protection with PEG20K. The present study is expected to improve the efficacy of the PEGylated Hb as an oxygen therapeutic.     

Site-specific PEGylation of hemoglobin at Cys-93(beta): correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain

Increasing the molecular size of acellular hemoglobin (Hb) has been proposed as an approach to reduce its undesirable vasoactive properties. The finding that bovine Hb surface decorated with about 10 copies of PEG5K per tetramer is vasoactive provides support for this concept. The PEGylated bovine Hb has a strikingly larger molecular radius than HbA (1). The colligative properties of the PEGylated bovine Hb are distinct from those of HbA and even polymerized Hb, suggesting a role for the colligative properties of PEGylated Hb in neutralizing the vasoactivity of acellular Hb. To correlate the colligative properties of surface-decorated Hb with the mass of the PEG attached and also its vasoactivity, we have developed a new maleimide-based protocol for the site-specific conjugation of PEG to Hb, taking advantage of the unusually high reactivity of Cys-93(beta) of oxy HbA and the high reactivity of the maleimide to protein thiols. PEG chains of 5, 10, and 20 kDa have been functionalized at one of their hydroxyl groups with a maleidophenyl moiety through a carbamate linkage and used to conjugate the PEG chains at the beta-93 Cys of HbA to generate PEGylated Hbs carrying two copies of PEG (of varying chain length) per tetramer. Homogeneous preparations of (SP-PEG5K)(2)-HbA, (SP-PEG10K)(2)-HbA, and (SP-PEG20K)(2)-HbA have been isolated by ion exchange chromatography. The oxygen affinity of Hb is increased slightly on PEGylation, but the length of the PEG-chain had very little additional influence on the O(2) affinity. Both the hydrodynamic volume and the molecular radius of the Hb increased on surface decoration with PEG and exhibited a linear correlation with the mass of the PEG chain attached. On the other hand, both the viscosity and the colloidal osmotic pressure (COP) of the PEGylated Hbs exhibited an exponential increase with the increase in PEG chain length. In contrast to the molecular volume, viscosity, and COP, the vasoactivity of the PEGylated Hbs was not a direct correlate of the PEG chain length. There appeared to be a threshold for the PEG chain length beyond which the protection against vasoactivity is decreased. These results suggest that the modulation of the vasoactivity of Hb by PEG could be a function of the surface shielding afforded by the PEG, the latter being a function of the disposition of the PEG chain on the protein surface, which in turn is a function of the length of the PEG chain. Thus, the biochemically homogeneous PEGylated Hbs described in the present study, surface-decorated with PEG chains of appropriate size, could serve as potential candidates for Hb-based oxygen carriers.     

PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.     

Rational surface engineering of an arginine deiminase (an antitumor enzyme) for increased PEGylation efficiency

Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, its application as anticancer drug is hampered by its poor stability under physiological conditions in the bloodstream. Commonly, random PEGylation is being used for increasing the stability of ADI and in turn the improved half-life. However, the traditional random PEGylation usually leads to poor PEGylation efficiency due to the limited number of Lys on the protein surface. To boost the PEGylation efficiency and enhance the stability of ADI further, surface engineering of PpADI (an ADI from Pseudomonas plecoglossicida) to increase the suitable PEGylation sites was carried out. A new in silico approach for increasing the PEGylation sites was developed. The validation of this approach was performed on previously identified PpADI variant M31 to increase potential PEGylation sites. Four Arg residues on the surface of PpADI M31 were selected through three criteria and subsequently substituted to Lys, aiming for providing primary amines for PEGylation. Two out of the four substitutions (R299K and R382K) enhanced the stability of PEGylated PpADI in human serum. The average numbers of PEGylation sites were increased from ~12 (tetrameric PpADI M31, starting point) to ~20 (tetrameric PpADI M36, final variant). Importantly, the PEGylated PpADI M36 after PEGylation exhibited significantly improved T_m values (M31: 40°C; M36: 40°C; polyethylene glycol [PEG]-M31: 54°C; PEG-M36: 64°C) and half-life in human serum (M31: 1.9 days; M36: 2.0 days; PEG-M31: 3.2 days; PEG-M36: 4.8 days). These proved that surface engineering is an effective approach to increase the PEGylation efficiency which therefore enhances the stability of therapeutic enzymes. Furthermore, the PEGylated PpADI M36 represents a highly attractive candidate for the treatment of arginine-auxotrophic tumors.     

Use of oligonucleotides to define the site of interstrand cross-links induced by Adriamycin.

It has been known for several years that Adriamycin forms adducts and interstrand cross-links when reacted for long periods of time with bacterial and mammalian DNA in vitro, with the cross-link being restricted to 2 bp elements containing GpC sequences. The self-complementary 20mer deoxyoligonucleotide TA4T4GCA4T4A has been used in this study as a model of the apparent G-G cross-linking site at GpC sequences. The rate of formation of cross-links, as well as the dependence on both Adriamycin and Fe(III) concentration, were similar with this oligonucleotide as compared with calf thymus DNA. The cross-linking was demonstrated on both denaturing and non-denaturing sequencing gels. The half-life of the G-G cross-link was 40 h, consistent with that implied with high molecular weight, heterogeneous sequence DNA. Exonuclease III digests of adducts formed with 20mer deoxyoligonucleotides containing single, central G-G, G-I and I-I potential cross-links revealed that a guanine residue is required at both ends of the cross-link. No cross-linking was observed with a similar oligonucleotide containing only a single central (G.C) bp.     

Combining the influence of two low O2 affinity-inducing chemical modifications of the central cavity of hemoglobin

HexaPEGylated hemoglobin (Hb), a non-hypertensive Hb, exhibits high O2 affinity, which makes it difficult for it to deliver the desired levels of oxygen to tissues. The PEGylation of very low O2 affinity Hbs is now contemplated as the strategy to generate PEGylated Hbs with intermediate levels of O2 affinity. Toward this goal, a doubly modified Hb with very low O2 affinity has been generated. The amino terminal of the beta-chain of HbA is modified by 2-hydroxy, 3-phospho propylation first to generate a low oxygen affinity Hb, HPPr-HbA. The oxygen affinity of this Hb is insensitive to DPG and IHP. Molecular modeling studies indicated potential interactions between the covalently linked phosphate group and Lys-82 of the trans beta-chain. To further modulate the oxygen affinity of Hb, the alpha alpha-fumaryl cross-bridge has been introduced into HPPr-HbA in the mid central cavity. The doubly modified HbA (alpha alpha-fumaryl-HPPr-HbA) exhibits an O2 affinity lower than that of either of the singly modified Hbs, with a partial additivity of the two modifications. The geminate recombination and the visible resonance Raman spectra of the photoproduct of alpha alpha-fumaryl-HPPr-HbA also reflect a degree of additive influence of each of these modifications. The two modifications induced a synergistic influence on the chemical reactivity of Cys-93(beta). It is suggested that the doubly modified Hb has accessed the low affinity T-state that is non-responsive to effectors. The doubly modified Hb is considered as a potential candidate for generating PEGylated Hbs with an O2 affinity comparable to that of erythrocytes for developing blood substitutes.     

Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration

Blood transfusion is still a critical therapy in many diseases, traumatic events and war battlefields. However, blood cross-matching and storage may limit its applicability, especially in Third World countries. Moreover, haemoglobin, which in red blood cells is the key player in the oxygen transport from lung to tissues, when free in the plasma causes hypertension and renal failure. This investigation was aimed at the development of a novel haemoglobin-based oxygen carrier with low vasoactivity and renal filtration properties. Human haemoglobin was chemically conjugated with polyethylene glycol (PEG) under either aerobic or anaerobic conditions, following different chemical procedures. The resulting PEGylated haemoglobin products were characterized in terms of oxygen affinity, cooperativity, effects of protons and carbon dioxide concentration, and oxidation stability, and were transfused into rats to evaluate vasoactivity and renal filtration. A deoxyhaemoglobin, conjugated with seven PEG and seven propionyl groups, which we called Euro-PEG-Hb, did not produce profound hypertension, was 99% retained within 6 h, and exhibited oxygen binding properties and allosteric effects more similar to human haemoglobin A than the other tested PEGylated haemoglobin derivatives, thus appearing a very promising candidate as blood substitute.     

Analysis of functionalization of methoxy-PEG as maleimide-PEG

The design of the extension arm-facilitated PEGylation (EAFP) of proteins takes advantage of the high selective and quantitative aspects of the thiol-maleimide reaction. However, the efficiency of EAFP with hemoglobin varied with the batches of maleimide-PEG. The low level of functionalization of monomethoxy-PEG (mPEG) as maleimide-PEG has been now investigated as the potential source of this variation. New chemical approaches for the estimation of the functionalization of mPEG using the reaction of the thiol groups of glutathione, dithiothreitol, and hemoglobin with maleimide-PEG have been developed. The single-step modular approach to the synthesis of maleimidophenyl-PEG (MPPEG) that involved the condensation of p-maleimidophenyl isocyanate with mPEG has been optimized to generate a product with an overall purity of 80%. The NMR approach correlates well with the estimates made by the new chemical approaches. Commercial maleimide-PEG reagents synthesized using multiple steps exhibited a lower level of functionalization as reflected by these chemical estimations. The better functionalization of MPPEG increases the efficiency of EAFP as reflected by the generation of hexaPEGylated Hb and the masking of the D antigen of RBCs. This new EAFP protocol is expected to improve the cost effectiveness of the generation of hexaPEGylated Hb, PEGylated albumin, and PEGylated RBCs as new PEGylated therapeutics.     

Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).     

Review: Ontogeny of the oxygen transport in the blood]

The biological importance of the oxygen affinity of the tetrameric haemoglobin molecule and its dependency from co-factors is described. Oxygen affinity variations during development from embryonic to adult values are discussed in respect to oxygen uptake from maternal blood via placenta, in lungs and gills as well as to oxygen delivery into tissues. Comparative aspects of the molecular mechanisms leading to affinity changes are presented.     

Di-isodityrosine is the intermolecular cross-link of isodityrosine-rich extensin analogs cross-linked in vitro

Extensins are cell wall hydroxyproline-rich glycoproteins that form covalent networks putatively involving tyrosyl and lysyl residues in cross-links catalyzed by one or more extensin peroxidases. The precise cross-links remain to be chemically identified both as network components in muro and as enzymic products generated in vitro with native extensin monomers as substrates. However, some extensin monomers contain variations within their putative cross-linking motifs that complicate cross-link identification. Other simpler extensins are recalcitrant to isolation including the ubiquitous P3-type extensin whose major repetitive motif, Hyp)(4)-Ser-Hyp-Ser-(Hyp)(4)-Tyr-Tyr-Tyr-Lys, is of particular interest, not least because its Tyr-Tyr-Tyr intramolecular isodityrosine cross-link motifs are also putative candidates for further intermolecular cross-linking to form di-isodityrosine. Therefore, we designed a set of extensin analogs encoding tandem repeats of the P3 motif, including Tyr --> Phe and Lys --> Leu variations. Expression of these P3 analogs in Nicotiana tabacum cells yielded glycoproteins with virtually all Pro residues hydroxylated and subsequently arabinosylated and with likely galactosylated Ser residues. This was consistent with earlier analyses of P3 glycopeptides isolated from cell wall digests and the predictions of the Hyp contiguity hypothesis. The tyrosine-rich P3 analogs also contained isodityrosine, formed in vivo. Significantly, these isodityrosine-containing analogs were further cross-linked in vitro by an extensin peroxidase to form the tetra-tyrosine intermolecular cross-link amino acid di-isodityrosine. This is the first identification of an inter-molecular cross-link amino acid in an extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking extensins in muro.     

Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins

Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys(185)-Lys(220) cross-link, which is unique to the PrPβ and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.     

Identification of glucose-derived cross-linking sites in ribonuclease A

The accumulation of glycation derived cross-links has been widely implicated in extracellular matrix damage in aging and diabetes, yet little information is available on the cross-linking sites in proteins and the intra- versus intermolecular character of cross-linking. Recently, glucosepane, a 7-membered heterocycle formed between lysine and arginine residues, has been found to be the single major cross-link known so far to accumulate during aging. As an approach toward identification of glucose derived cross-linking sites, we have preglycated ribonuclease A first for for 14 days with 500 mM glucose, followed by a 4-week incubation in absence of glucose. MALDI-TOF analysis of tryptic digests revealed the presence of Amadori products (Delta m/ z = 162) at K1, K7, K37 and K41, in accordance with previous studies. In addition, K66, K98 and K104 were also modified by Amadori products. Intramolecular glucosepane cross-links were observed at K41-R39 and K98-R85. Surprisingly, the only intermolecular cross-link observed was the 3-deoxyglucosone-derived DODIC at K1-R39. The identity of cross-linked peptides was confirmed by sequencing with tandem mass spectrometry. Recombinant ribonuclease A mutants R39A, R85A, and K91A were produced, purified, and glycated to further confirm the importance of these sites on protein cross-linking. These data provide the first documentation that both intramolecular and intermolecular cross-links form in glucose-incubated proteins.     

Molecular Insight into the Steric Shielding Effect of PEG on the Conjugated Staphylokinase: Biochemical Characterization and Molecular Dynamics Simulation

PEGylation is a successful approach to improve potency of a therapeutic protein. The improved therapeutic potency is mainly due to the steric shielding effect of PEG. However, the underlying mechanism of this effect on the protein is not well understood, especially on the protein interaction with its high molecular weight substrate or receptor. Here, experimental study and molecular dynamics simulation were used to provide molecular insight into the interaction between the PEGylated protein and its receptor. Staphylokinase (Sak), a therapeutic protein for coronary thrombolysis, was used as a model protein. Four PEGylated Saks were prepared by site-specific conjugation of 5 kDa/20 kDa PEG to N-terminus and C-terminus of Sak, respectively. Experimental study suggests that the native conformation of Sak is essentially not altered by PEGylation. In contrast, the bioactivity, the hydrodynamic volume and the molecular symmetric shape of the PEGylated Sak are altered and dependent on the PEG chain length and the PEGylation site. Molecular modeling of the PEGylated Saks suggests that the PEG chain remains highly flexible and can form a distinctive hydrated layer, thereby resulting in the steric shielding effect of PEG. Docking analyses indicate that the binding affinity of Sak to its receptor is dependent on the PEG chain length and the PEGylation site. Computational simulation results explain experimental data well. Our present study clarifies molecular details of PEG chain on protein surface and may be essential to the rational design, fabrication and clinical application of PEGylated proteins.