kDa mPEG Research Articles

Phase 1/2 dose escalation and dose expansion study of TransCon IL-2 β/γ alone or in combination with pembrolizumab: Initial results from dose escalation.

e14597 Background: TransCon IL-2 β/γ is a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog (IL-2 β/γ) designed to optimally address the drawbacks of interleukin-2 (IL-2) therapy. IL-2 β/γ was created by permanently attaching a 5 kDa mPEG in the IL-2Rα binding site. To improve the pharmacokinetic (PK) properties, IL-2 β/γ was transiently attached to a 40 kDa mPEG carrier via a TransCon linker creating a prodrug, TransCon IL-2 β/γ. Under physiological conditions, TransCon IL-2 β/γ releases active IL-2 β/γ from the carrier via autocleavage in a controlled manner. The associated long effective half-life and sustained IL-2 β/γ exposure results in robust activation of IL-Rβ/γ+ cytotoxic immune cells, at a lower Cmax compared to IL-2 therapy. The primary objectives of the IL-Believe trial are to evaluate safety and tolerability, and to define the maximum tolerated dose and recommended Phase 2 dose of TransCon IL-2 β/γ alone or in combination with pembrolizumab. Methods: In dose escalation (3+3 design), patients aged ≥18 with advanced solid tumors receive TransCon IL-2 β/γ as monotherapy or in combination with pembrolizumab with IV dosing every 3 weeks. Disease is assessed every 9 weeks using RECIST 1.1. Safety, PK, and pharmacodynamics (PD) are evaluated. Results: As of 25 Jan 2023, 27 patients were treated in dose escalation: 18 in monotherapy (Dose Levels (DL) 1-4) and 9 in combination therapy (DL1, DL2). The most common tumor types in monotherapy and in combination therapy were head & neck (19%), colorectal (11%) and endometrial (11%). Patients received a median of 3 prior lines of therapy in monotherapy. The most common Treatment Related Adverse Events (TRAEs) in monotherapy and in combination therapy were pyrexia (30%), Cytokine Release Syndrome (CRS) (26%) and fatigue (22%). Grade 3 (G3) TRAEs in monotherapy were anemia (17%), CRS (6%), thrombocytopenia (6%), hypoxia (6%), all reported at DL4. 1 Dose Limiting Toxicity of G3 CRS was reported at DL4. Preliminary PK results showed systemic drug exposure of TransCon IL-2 β/γ with a half-life of ~ 35 hours and no PK interaction with pembrolizumab was observed. PD data were consistent with sustained activation and dose dependent expansion of cytotoxic effector cells, in comparison, Tregs were only expanded minimally. Absolute lymphocyte counts increased in a dose dependent manner while eosinophils remained low. Prolonged stable disease ( > 32 weeks) was observed in one pancreatic cancer patient on monotherapy. Conclusions: TransCon IL-2 β/γ is generally well-tolerated as monotherapy and in combination with pembrolizumab. The pharmacodynamics data confirm a durable activation and expansion of IL-2Rβ/γ+ cytotoxic immune cells over IL-2Rα+ eosinophils and Tregs. Clinical trial information: NCT05081609 .

Engineering an Fc-Fusion of a Capsule Degrading Enzyme for the Treatment of Anthrax.

Polymers of d-glutamic acid (PDGA) form the capsule of the highly virulent Ames strain of B.anthracis. PDGA is antiphagocytic and weakly immunogenic; it enables the bacteria to evade the innate immune responses. CapD is an enzyme that catalyzes the covalent anchoring of PDGA. CapD is an Ntn-amido hydrolase that utilizes an internal Thr-352 as its nucleophile and general acid and base. An internal cleavage produces a free N-terminal Thr-352 and a short and long polypeptide chain. The chains were circularly permuted (CP) to move Thr-352 to the N-terminus of the polypeptide. We previously showed that a branched PEG-CapDS334C-CP could protect mice (80% survival) against a 5 LD50 challenge with B.anthracis Ames without the use of antibiotics, monoclonals, or vaccines. In attempts to improve the in vivo circulation time of CapD and enhance its avidity to its polymeric substrate, an Fc-domain of a mouse IgG1 was fused to CapDS334C-CP and the linker length and sequence were optimized. The resulting construct, Fc-CapDS334C-CP, then was pegylated with a linear 2 kDa mPEG at S334C to produce mPEG-Fc-CapDS334C-CP. Interestingly, the fusion of the Fc-domain and incorporation of the S334C mutation imparted acid stability, but slightly reduced the kcat (∼ 2-fold lower). In vivo, the measured protein concentration in sera was higher for the Fc-fusion constructs compared to the mPEG-Fc-CapDS334C-CP. However, the exposure calculated from measured sera enzymatic activity was higher for the mPEG-CapDS334C-CP. The pegylated Fc-fusion was less active than the PEG-CapDS334C-CP, but detectable in sera at 24 h by immunoblot. Here we describe the engineering of a soluble, active, pegylated Fc-fusion of B.anthracis CapD (mPEG-Fc-CapD-CP) with activity in vitro, in serum, and on encapsulated bacteria.

TransCon IL-2 β/γ: a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 variant with improved pharmacokinetics and potent activation of cytotoxic immune cells for the treatment of cancer

BackgroundRecombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe toxicities including cytokine storm and vascular leak syndrome (VLS). IL-2 promotes antitumor function of IL-2Rβ/γ+ natural killer (NK)...

Enhanced Bioactivity of a Human GHR Antagonist Generated by Solid-Phase Site-Specific PEGylation.

Growth hormone (GH) has been implicated in cancer progression andis a potential target for anticancer therapy. Currently, pegvisomant is the only GH receptor (GHR) antagonist approved for clinical use. Pegvisomant is a mutated GH molecule (B2036) which is PEGylated on amine groups to extend serum half-life. However, PEGylation significantly reduces the bioactivity of the antagonist in mice. To improve bioactivity, we generated a series of B2036 conjugates with the site-specific attachment of 20, 30, or 40 kDa methoxyPEG maleimide (mPEG maleimide) by introduction of a cysteine residue at amino acid 144 (S144C). Recombinant B2036-S144C was expressed in Escherichia coli, purified, and then PEGylated using cysteine-specific conjugation chemistry. To avoid issues with dimerization due to the introduced cysteine, B2036-S144C was PEGylated while immobilized on an Ni-nitrilotriacetic (Ni-NTA) acid column, which effectively reduced disulfide-mediated dimer formation and allowed efficient conjugation to mPEG maleimide. Following PEGylation, the IC50 values for the 20, 30, and 40 kDa mPEG maleimide B2036-S144C conjugates were 66.2 ± 3.8, 106.1 ± 7.1, and 127.4 ± 3.6 nM, respectively. The circulating half-life of the 40 kDa mPEG conjugate was 58.3 h in mice. Subcutaneous administration of the 40 kDa mPEG conjugate (10 mg/kg/day) reduced serum insulin-like growth factor I (IGF-I) concentrations by 50.6%. This in vivo reduction in serum IGF-I was at a considerably lower dose compared to the higher doses required to observe comparable activity in studies with pegvisomant. In conclusion, we have generated a novel PEGylated GHR antagonist by the solid-phase site-specific attachment of mPEG maleimide at an introduced cysteine residue, which effectively reduces serum IGF-I in vivo.

Effect of Gold Nanoparticle Size on Their Properties as Contrast Agents for Computed Tomography

Computed tomography (CT) is one of the most commonly used clinical imaging modalities. There have recently been many reports of novel contrast agents for CT imaging. In particular, the development of gold nanoparticles (AuNP) as CT contrast agents is a topic of intense interest. AuNP have favorable characteristics for this application such as high payloads of contrast generating material, strong X-ray attenuation, excellent biocompatibility, tailorable surface chemistry, and tunable sizes and shapes. However, there have been conflicting reports on the role of AuNP size on their contrast generation for CT. We therefore sought to extensively investigate the AuNP size-CT contrast relationship. In order to do this, we synthesized AuNP with sizes ranging from 4 to 152 nm and capped them with 5 kDa m-PEG. The contrast generation of AuNP of different sizes was investigated with three clinical CT, a spectral photon counting CT (SPCCT) and two micro CT systems. X-ray attenuation was quantified as attenuation rate in Hounsfield units per unit concentration (HU/mM). No statistically significant difference in CT contrast generation was found among different AuNP sizes via phantom imaging with any of the systems tested. Furthermore, in vivo imaging was performed in mice to provide insight into the effect of AuNP size on animal biodistribution at CT dose levels, which has not previously been explored. Both in vivo imaging and ex vivo analysis with inductively coupled plasma optical emission spectroscopy (ICP-OES) indicated that AuNP that are 15 nm or smaller have long blood circulation times, while larger AuNP accumulated in the liver and spleen more rapidly. Therefore, while we observed no AuNP size effect on CT contrast generation, there is a significant effect of size on AuNP diagnostic utility.

Conjugation to 10 kDa Linear PEG Extends Serum Half-Life and Preserves the Receptor-Binding Ability of mmTRAIL with Minimal Stimulation of PEG-Specific Antibodies.

The poor in vivo potencies of most therapeutic proteins might be attributed to their short serum half-lives. PEGylation is a well-established method and has been clinically proven to improve pharmacokinetics. mmTRAIL exhibited supercytotoxicity in a variety of tumor cells, but its serum half-life was less than 10 min in mice. Here, mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL-20K were produced by N-terminus-specific PEGylation of mmTRAIL with 5, 10, or 20 kDa mPEG, respectively. The particle sizes of mmTRAIL-5K, mmTRAIL-10K, and mmTRAIL-20K were 9.09 ± 2.76, 12.62 ± 4.05, and 15.68 ± 4.95 nm, which were higher than the threshold (∼7 nm) of renal clearance. Accordingly, mmTRAIL-5K exhibited a serum half-life of 30 min only 3 times longer than that of mmTRAIL. However, both mmTRAIL-10K and mmTRAIL-20K exhibited similar serum half-lives ranging from 350 to 400 min, indicating that PEGylation with 10 or 20 kDa mPEG significantly improved the pharmacokinetics of mmTRAIL. However, death receptor binding of mmTRAIL-20K was reduced 5- to 8-fold, resulting in a 3-fold reduction of cytotoxicity. Additionally, repeated administration of mmTRAIL-20K elicited both mPEG-specific IgG and IgM antibody responses in rats. In contrast, the receptor binding and cytotoxicity of mmTRAIL-10K were similar to those of mmTRAIL. Repeated administration of mmTRAIL-10K did not obviously stimulate mPEG-specific antibody responses in rats and rhesus monkeys. Of the three PEGylated mmTRAIL analogues, mmTRAIL-10K exerted the greatest tumor suppression in mice bearing human tumor xenografts. These results demonstrated that conjugation of mmTRAIL to 10 kDa mPEG was better than that to 5 or 20 kDa mPEG for enhancing antitumor effects.

Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients

IntroductionPegloticase, a PEGylated recombinant porcine uricase, is approved for treating refractory gout at a dose of 8 mg intravenous (IV) every 2 weeks. However, during phase 1 testing, pharmacokinetics supported less frequent dosing. Also, single doses of pegloticase unexpectedly induced antibodies (Ab) that bound to polyethylene glycol (PEG). We have conducted a phase 2 trial to evaluate every 3-week dosing, and to further define the Ab response to pegloticase. Organ transplant recipients were included, as they are prone to severe gout that is difficult to manage, and because treatment to prevent graft rejection might influence the immune response to pegloticase.MethodsPlasma uricase activity (pUox), urate concentration (pUA), and clinical response were monitored during up to 5 infusions in 30 patients, including 7 organ transplant recipients. Depending on whether pUA <6 mg/dL was achieved and maintained, patients were classified as non (NR), persistent (PR), or transient (TR) responders. Ab to pegloticase and 10 kDa mPEG were monitored by enzyme linked immunosorbent assay and specificity was further defined.ResultsWe observed 17 PR, 12 TR, and 1 NR; 21 patients (16 PR, 5 TR) received all 5 infusions. Over the 15-week trial, pUA in PR averaged 1.0 ± 0.4 mg/dL; T½ for pUox was approximately 13 days, and area under the curve after dose 5 was approximately 30% higher than after dose 1. PR showed clinical benefit and in some, tophi resolved. In 11 of 12 TR, pUox fell rapidly and hyperuricemia recurred before dose 2. In all TR and NR, loss of response to pegloticase was accompanied by Ab to PEG, which was pre-existing in half of those who had no prior exposure to pegloticase. No PR, and 1 one out of 7 organ transplant recipients, had a sustained Ab response to pegloticase.ConclusionsEvery 3-week dosing is effective and may enhance the utility of pegloticase for treating refractory gout. Ab to PEG, which were pre-existing or induced by treatment, caused rapid loss of efficacy and increased the risk of infusion reactions. Organ transplant recipients can benefit from pegloticase, and may be less prone than non-recipients to developing anti-PEG Ab. Investigation of immunosuppressive strategies to minimize anti-PEG Ab is warranted.Trial registrationClincalTrials.gov identifier: NCT00111657

PEGylation Extends Circulation Half-Life While Preserving In Vitro and In Vivo Activity of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)

Excess proteolytic activity of matrix metalloproteinases (MMPs) contributes to the development of arthritis, cardiovascular diseases and cancer progression, implicating these enzymes as therapeutic targets. While many small molecule inhibitors of MMPs have been developed, clinical uses have been limited, in part by toxicity and off-target effects. Development of the endogenous tissue inhibitors of metalloproteinases (TIMPs) as recombinant biopharmaceuticals represents an alternative therapeutic approach; however, the short plasma half-life of recombinant TIMPs has restricted their potential in this arena. To overcome this limitation, we have modified recombinant human TIMP-1 (rhTIMP-1) by PEGylation on lysine residues. We analyzed a mixture of mono- and di-PEGylated rhTIMP-1 species modified by attachment of 20 kDa mPEG chains (PEG20K-TIMP-1), as confirmed by SELDI-TOF mass spectrometry. This preparation retained complete inhibitory activity toward the MMP-3 catalytic domain and partial inhibitory activity toward full length MMP-9. Pharmacokinetic evaluation showed that PEGylation extended the plasma half-life of rhTIMP-1 in mice from 1.1 h to 28 h. In biological assays, PEG20K-TIMP-1 inhibited both MMP-dependent cancer cell invasion and tumor cell associated gelatinase activity. Overall these results suggest that PEGylated TIMP-1 exhibits improved potential for development as an anti-cancer recombinant protein therapeutic, and additionally may offer potential for clinical applications in the treatment of other diseases.

Characterization, efficacy, pharmacokinetics, and biodistribution of 5 kDa mPEG modified tetrameric canine uricase variant

PEGylated uricase is a promising anti-gout drug, but the only commercially marketed 10kDa mPEG modified porcine-like uricase (Pegloticase) can only be used for intravenous infusion. In this study, tetrameric canine uricase variant was modified by covalent conjugation of all accessible ɛ amino sites of lysine residues with a smaller 5kDa mPEG (mPEG-UHC). The average modification degree and PEGylation homogeneity were evaluated. Approximately 9.4 5kDa mPEG chains were coupled to each monomeric uricase and the main conjugates contained 7–11 mPEG chains per subunit. mPEG-UHC showed significantly therapeutic or preventive effect on uric acid nephropathy and acute urate arthritis based on three different animal models. The clearance rate from an intravenous injection of mPEG-UHC varied significantly between species, at 2.61mL/h/kg for rats and 0.21mL/h/kg for monkeys. The long elimination half-life of mPEG-UHC in non-human primate (191.48h, intravenous injection) indicated the long-term effects in humans. Moreover, the acceptable bioavailability of mPEG-UHC after subcutaneous administration in monkeys (94.21%) suggested that subcutaneous injection may be regarded as a candidate administration route in clinical trails. Non-specific tissue distribution was observed after administration of 125I-labeled mPEG-UHC in rats, and elimination by the kidneys into the urine is the primary excretion route.

PEGylation is effective in reducing immunogenicity, immunotoxicity, and hepatotoxicity of α-momorcharin in vivo

Background and aim: α-momorcharin (α-MMC), a type I ribosome-inactivating protein (RIP) from Momordica charantia, is well known for its antitumor and antivirus activities. However, the immunotoxicity and hepatotoxicity hampers its potential therapeutic usage. In order to reduce its toxicity, we had modified the α-MMC with polyethylene glycol (PEG), and detected the toxicity of the PEGylated α-MMC conjugates (α-MMC-PEG) in vivo.Materials and methods: After α-MMC purified from bitter melon seeds, α-MMC-PEG was constructed with a branched 20 kDa (mPEG) 2-Lys-NHS, the tests of immunogenicity, immunotoxicity, and general toxicity of α-MMC-PEG were conducted in guinea pig and rat.Results: The titer of specific IgG in rats, immunized by α-MMC-PEG, were approximately one-third of those that by α-MMC, all the guinea pigs treated with α-MMC died of anaphylaxis shock within 5 min, while no animals treated with α-MMC-PEG died in the active systemic anaphylaxis (ASA) test. The passive cutaneous anaphylaxis (PCA) reaction of α-MMC-PEG challenge in rats was significantly smaller than that of the α-MMC. The liver damage was greatly released, such as the change of globulin (GLB), aspartate aminotransferase (AST), total bilirubin (TBIL) cholesterol (CHOL), albumin (ALB), and the degree of hepatocyte necrosis in repeated toxicity study.Conclusions: PEGylation is effective in reducing the immunogenicity, immunotoxicity, and hepatotoxicity of α-MMC in vivo.

Role of the Methoxy Group in Immune Responses to mPEG-Protein Conjugates

Anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins and liposomes, all of which contain methoxyPEG (mPEG). The goal of this research was to assess the role of the methoxy group in the immune responses to mPEG conjugates and the potential advantages of replacing mPEG with hydroxyPEG (HO-PEG). Rabbits were immunized with mPEG, HO-PEG, or t-butoxyPEG (t-BuO-PEG) conjugates of human serum albumin, human interferon-α, or porcine uricase as adjuvant emulsions. Assay plates for enzyme-linked immunosorbent assays (ELISAs) were coated with mPEG, HO-PEG, or t-BuO-PEG conjugates of the non-cross-reacting protein, porcine superoxide dismutase (SOD). In sera from rabbits immunized with HO-PEG conjugates of interferon-α or uricase, the ratio of titers of anti-PEG antibodies detected on mPEG-SOD over HO-PEG-SOD (“relative titer”) had a median of 1.1 (range 0.9–1.5). In contrast, sera from rabbits immunized with mPEG conjugates of three proteins had relative titers with a median of 3.0 (range 1.1–20). Analyses of sera from rabbits immunized with t-BuO-PEG-albumin showed that t-butoxy groups are more immunogenic than methoxy groups. Adding Tween 20 or Tween 80 to buffers used to wash the assay plates, as is often done in ELISAs, greatly reduced the sensitivity of detection of anti-PEG antibodies. Competitive ELISAs revealed that the affinities of antibodies raised against mPEG-uricase were c. 70 times higher for 10 kDa mPEG than for 10 kDa PEG diol and that anti-PEG antibodies raised against mPEG conjugates of three proteins had >1000 times higher affinities for albumin conjugates with c. 20 mPEGs than for analogous HO-PEG-albumin conjugates. Overall, these results are consistent with the hypothesis that antibodies with high affinity for methoxy groups contribute to the loss of efficacy of mPEG conjugates, especially if multiply-PEGylated. Using monofunctionally activated HO-PEG instead of mPEG in preparing conjugates for clinical use might decrease this undesirable effect.

PEGylation of alpha-momorcharin: synthesis and characterization of novel anti-tumor conjugates with therapeutic potential

Alpha-momorcharin (alpha-MMC) is a ribosome-inactivating protein (RIP) with excellent cytotoxicity to tumor cells. However, its strong immunogenicity and short plasma half-life limit its clinical applications. To overcome this, we have to PEGylated alpha-MMC using a branched 20 kDa (mPEG) (2)-Lys-NHS. Homogeneous mono-, di- and tri-PEGylated alpha-MMCs were synthesized, purified and characterized. In vitro and in vivo analysis indicated that the serial PEG-conjugates preserved moderate anti-tumor activity with 36% acute toxicity and at most 66% immunogenicity decrease. These results suggested the potential application of alpha-MMC-PEG conjugates as an anti-tumor agent.

The effect of grafted methoxypoly(ethylene glycol) chain length on the inhibition of respiratory syncytial virus (RSV) infection and proliferation

Respiratory syncytial virus (RSV) is a significant cause of morbidity in humans. To date, no effective treatments exist and current prophylactic therapy access is limited and is only ∼50% effective. To attenuate the risk of RSV infection, we hypothesized that bioengineering of either the virus particle or host cell via the covalent grafting of methoxypoly(ethylene glycol) [mPEG] would prevent infection. To this end, the anti-viral effects of grafting concentration, linker chemistry and polymer length on RSV infection was assessed. For viral modification, short chain polymers (2 kDa) were significantly more effective than long chain (20 kDa) polymers. In contrast, modification of host cells with small polymers provided no (∼0%) protection while long chain polymers effectively prevented infection. For example, at 48 hours post-infection at a multiplicity of infection of 0.5 and grafting concentrations of 5, 7.5, and 15 m m, 20 kDa mPEG decreased infection by 45, 83, and 91%, respectively. Importantly, both viral and host cell PEGylation strategies were able to provide near complete protection against RSV infection of both non-polarized and polarized cells. In conclusion, mPEG-modification of either RSV or the host cell is a highly effective prophylactic strategy for preventing viral infection.

Anti-tumor activity and immunological modification of ribosome-inactivating protein (RIP) from &amp;lt;italic&amp;gt;Momordica charantia&amp;lt;/italic&amp;gt; by covalent attachment of polyethylene glycol

Ribosome-inactivating proteins (RIPs) are a family of enzymes that depurinate rRNA and inhibit protein biosynthesis. Here we report the purification, apoptosis-inducing activity, and polyethylene glycol (PEG) modification of RIP from the bitter melon seeds. The protein has a homogenous N-terminal sequence of NAsp- Val-Ser-Phe-Arg. Moreover, the RIP displayed strong apoptosis-inducing activity and suppressed cancer cell growth. This might be attributed to the activation of caspases-3. To make it available for in vivo application, the immunogenicity of RIP was reduced by chemical modification with 20 kDa (mPEG)(2)-Lys-NHS. The inhibition activity of both PEGylated and non-PEGylated RIP against cancer cells was much stronger than against normal cells, and the antigenicity of PEGylated RIP was reduced significantly. Our results suggested that the PEGylated RIP might be potentially developed as anti-cancer drug.

Solid-Phase PEGylation of Recombinant Interferon α-2a for Site-Specific Modification: Process Performance, Characterization, andin VitroBioactivity

'Solid-phase' PEGylation, in which a conjugation reaction attaches proteins to a solid matrix, has distinct advantages over the conventional, solution-phase process. We report a case study in which recombinant interferon (rhIFN) alpha-2a was adsorbed to a cation-exchange resin and PEGylated at the N-terminus by 5, 10, and 20 kDa mPEG aldehydes through reductive alkylation. After PEGylation, a salt gradient elution efficiently purified the mono-PEGylate of unwanted species such as unmodified IFN and unreacted PEG. Mono-PEGylation and purification were integrated into a single, chromatographic step. Depending on the molecular weight of the mPEG aldehyde, the mono-PEGylation yield ranged from 50 to 65%. Major problems associated with the solution-phase process such as random or uncontrollable multi-PEGylation and post-PEGylation purification difficulties were overcome. N-terminus sequencing and MALDI-TOF mass spectrophometry confirmed that the PEG molecule was conjugated only to the N-terminus. A cell proliferation study indicated reduced antiviral activity of the mono-PEGylate compared to that of the unmodified IFN. As higher molecular weight PEG was conjugated, in vitro bioactivity and antibody binding activity, as measured by a surface plasmon resonance biosensor, decreased. Nevertheless, trypsin resistance and thermal stability were considerably improved .

Reactivity of free thiol groups in type-I inositol trisphosphate receptors

The IP3R (inositol 1,4,5-trisphosphate receptor) Ca2+-release channel is known to be sensitive to thiol redox state. The present study was undertaken to characterize the number and location of reactive thiol groups in the type-I IP3R. Using the fluorescent thiol-reactive compound monobromobimane we found that approx. 70% of the 60 cysteine residues in the type-I IP3R are maintained in the reduced state. The accessibility of these residues was assessed by covalently tagging the IP3R in membranes with a 5 kDa or 20 kDa MPEG [methoxypoly(ethylene glycol) maleimide]. MPEG reaction caused a shift in the mobility of IP3R on SDS/PAGE that was blocked by pretreatment of the membranes with dithiothreitol, N-ethylmaleimide, mersalyl or thimerosal, indicating that MPEG reactivity was specific to thiol groups on the IP3R. Trypsin cleavage of the type-I IP3R generates five defined domains. In cerebellum membranes, MPEG reacted over a 5 min interval with tryptic fragment I and fragment III, but not fragments II, IV or V. Fragment I appears as a doublet in cerebellum membranes, corresponding to the presence and absence of the SI splice site in this region (SI is a spliced domain corresponding to amino acids 318-332). Only the fragment I band corresponding to the SI(+) splice form shifted after reaction with MPEG. Expression of SI(+) and SI(-) spliced forms in COS cell microsomes confirmed this result. The MPEG-induced shift was not prevented when the cysteine residue present in the SI splice domain (C326A) or the remaining seven cysteine residues in fragment I were individually mutated. Of the combination mutations screened, only the mutation of C206/214/326A blocked MPEG reactivity in fragment I. We conclude that a set of highly reactive cysteine residues in fragment I are differentially accessible in the SI(+) and SI(-) splice variants of the type-I IP3R.