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Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required., Graphical abstract, Subcutaneous administration of mRNA formulated within LNPs results in measurable plasma exposure of a secreted protein, albeit with dose-limiting local inflammatory responses. Inclusion of a steroid prodrug in mRNA LNPs resulted in an increased level and duration of protein expression as well as improved tolerability of the LNP following subcutaneous administration.

The very evolutionarily conserved human carbonic anhydrase-related polypeptide (CA-RP VIII) lacks the carbon-dioxide hydration-activity, characteristic of the enzymatically active carbonic anhydrases. We have expressed HCA-RP VIII as a glutathione-S-transferase fusion protein (GST-HCA-RP VIII). The purified HCA-RP VIII showed a substantially higher apparent molecular weight by gel-filtration compared to the molecular weight calculated from the amino acid sequence, indicating a larger than expected Stoke's radius. Like other studied CA's, the protein unfolds through two transitions at increasing concentrations of guanidine hydrochloride. The far-UV CD spectra of HCA-RP VIII indicates a secondary structure similar to that of the catalytically active HCA II. The very high sequence identity between human and mouse CA-RP VIII (98%), might indicate that the function of the protein involves binding of another protein. However, an attempt to use the GST-HCA-RP VIII fusion protein to affinity purify a ligand was unsuccessful.

Ca(2+)-ATPase from rat skeletal-muscle sarcoplasmic-reticulum has been reported to be less stable in old animals. These changes were suggested to be due to age-related modifications in the membrane. In the present study, the guanidinium-chloride (GuHCl)-induced inactivation, and reactivation by dilution of the denaturant, of this enzyme have been investigated. The cooperativity of the inactivation-transition was found to increase when the membrane was dissolved in the non-ionic detergent polyoxyethylene 10 laurylether. There is an inactive unfolding intermediate in equilibrium with the native state at low concentrations of GuHCl. This intermediate can be reactivated, even after as long a time as 19 h. The stability of this intermediate is only slightly affected by detergent solubilization of the enzyme. Hence, the membrane probably only plays a minor role in stabilizing the intermediate.

The kinetics of the refolding of the enzyme, human carbonic anhydrase II (HCA II), at different temperatures, together with the Escherichia coli chaperonin GroEL, has been studied. The Arrhenius plots for the spontaneous, GroEL-assisted, and GroEL/ES-assisted refolding of HCA II show that the apparent activation energy (Ea) is lower in the presence of the chaperonin GroEL alone than for the spontaneous reaction, whereas the apparent activation energy for the GroEL/ES-assisted reaction is almost the same as for the spontaneous reaction (85, 46, and 72 kJ/mol, for the spontaneous, GroEL, and GroEL/ES-assisted reactions, respectively).

The carbonic anhydrase (CA)-like protein, CA VIII, lacks the typical carbon dioxide hydrase activity of the CA isozymes. However, the high degree of amino acid sequence similarity between the products of the mouse and the human CA VIII genes suggests an important biological function. We have attempted to investigate the function of this gene in mammalian development by conducting an in situ hybridization study on sagittal sections of mouse embryos at gestation days of 9.5-16.5 using a 35S-labelled riboprobe. Results indicate that this gene (called Car8 in mice) is expressed as early as day 9.5 in a variety of organs including liver, branchial arches, neuroepithelium and developing myocardium. Between days 10.5 and 12.5, it showed a widespread distribution of mRNA expression that became more restricted as development progressed. The level of expression of Car8 mRNA was relatively high in the brain, liver, lung, heart, gut, thymus and epithelium covering the head and the oronasal cavity.

The initial yield of reactivation of GuHCl denatured human carbonic anhydrase II does not change with temperature between 3 and 35 degrees C. At temperatures above 35 degrees C, the enzymatic activity is not stable, but decreases over time. If the bacterial chaperonin GroEL is present during reactivation, the initial yield is lower compared to the spontaneous reaction at temperatures of 35-50 degrees C. However, unlike the spontaneous reactivation, the enzymatic activity with time in the presence of GroEL. In the presence of GroEL, native HCA II incubated at elevated temperatures will rapidly loose enzymatic activity to the same value as during reactivation at that particular temperature; most of the activity will recover if the temperature is lowered when GroEL is present. It is evident that there is an equilibrium between an inactive intermediate of HCA II, probably bound to GroEL, and active enzyme. Furthermore, proline isomerization is part of the rate-limiting step of refolding even in the presence of GroEL, and it is very noteworthy that prolyl isomerase will influence the refolding of HCA II in the presence of GroEL.

There are three gene families that encode zinc metalloenzymes that catalyze the reversible hydration of CO2. The encoded enzymes are termed carbonic anhydrases (CAs). The CA isozymes have been purified from representatives of all types of organisms. Most CAs are strongly inhibited by aromatic sulfonamides. Several chromatographic and electrophoretic methods have been devised to determine binding constants for sulfonamides to CAs, and these compounds have been extensively used for, often single-step, affinity chromatographic separation of CAs from complex matrixes. The purification of different CA isozymes from different organisms is reviewed, as are methods for detection of CAs during chromatography and electrophoresis.

The presence of GroEL/13S during the refolding of human carbonic anhydrase II (pseudo-wild type) was found to increase the yield of active enzyme from 65 to 100%. This chaperone action on the enzyme could be obtained by adding GroEL alone, and the time-course in that case was only moderately slower than the spontaneous process. Truncated forms of carbonic anhydrase, in which N-terminal helices were removed, also served as protein substrates for GroEL/ES. This demonstrates that N-terminally located helices are not obligatory as recognition motifs.

The structure of the gene encoding carbonic anhydrase I (CA I) was determined for the pigtail macaque Macaca nemestrina. When the deduced amino-acid sequence was compared with those of five other primates, four non-primate mammals and a turtle, seven residues were found to be unique and invariant to all of the CA I sequences. A scheme is presented for the probable evolutionary order of the six polymorphic nucleotide changes found in the coding regions of the CA I locus of pigtail macaques.

SUMMARY The genes encoding carbonic anhydrase I (CA I) have been characterized for chimpanzee (Pan troglodytes) and gorilla (Gorilla gorilla). In addition, 44 nucleotides (nt) at the 5’ end of the noncoding first exon (exon la), which is unique to the erythroid CA I mRNA, together with 188 nt of the adjacent 5’ flanking regions, were sequenced for the corresponding positions of the CA I of orangutan, pigtail macaque, and squirrel monkey. When these 5’ flanking regions are compared, along with those published for human and mouse CA I, they were found to contain several conserved sequences that may bind factors involved in the erythroid-specific expression of CA I. Comparisons of the human, chimpanzee, and gorilla coding and noncoding CA Z sequences do not significantly deviate from a pattern of trichotomy for the evolutionary origins of these three hominoid species.

A stopped-flow apparatus with many potential applications is described, in which the vertical design makes operaton easier. In addition, the system has adjustable driving pistons that facilitate refilling of syringes and changing of reaction solutions without allowing air to enter the system. This means that the use of reservoir syringes is redundant. The driving pistons can easily be adjusted to withstand different pressures and temperatures and the O-rings are simple to change when worn out. The vertical stopped-flow system is also flexible, as syringes of different size can be used and are easily changed when, for instance, different dilution ratios are needed.

Obesity is now recognized as a rapidly increasing worldwide threat to health, largely as a result of causing diabetes. Thus, considerable efforts are underway in the pharmaceutical industry to find drugs to treat this condition. Target validation in various academic and industrial laboratories has revealed a number of potential molecular targets in fat cells or adipocytes. By definition, obesity is too much fat, and we here review efforts to treat obesity and, by proxy, diabetes by modulating the metabolic state of adipocytes.

The prevalence of obesity is increasing rapidly in most parts of the world and effective therapeutic drugs are urgently needed. The discovery of leptin in 1994 initiated a new understanding of adipose tissue function, and adipose tissue is now known to not only store and release fatty acids, but also to produce a wealth of factors that have an impact on the regulation of body weight and blood glucose homeostasis. Also, adipocytes express proteins that engage signalling pathways playing important roles in fuel substrate and energy metabolism. These proteins constitute a diverse array of adipose target candidates for the development of drugs to treat obesity. Some of these potential targets have been validated and are now in drug development stages, providing hope that the current obesity epidemic can be addressed by effective drug treatments in the near future.

Leporin B (1), a novel demethylated analogue of leporin A (2), was isolated from a taxonomically unidentified fungal strain as part of an effort to discover compounds with the ability to increase expression levels of the enzyme hexokinase II. The structure was determined by spectral methods, including 1D and 2D NMR, and HRMS. The relative stereochemistry was assigned by NOESY experiments and coupling constants.

Several proteins have been discovered that either catalyze slow protein-folding reactions or assist folding in the cell. Prolyl isomerase, which has been shown to accelerate rate-limiting cis-trans peptidyl-proline isomerization steps in the folding pathway, can also participate in the protein-folding process as a chaperone. This function is exerted on an early folding intermediate of carbonic anhydrase, which is thereby prevented from aggregating, whereas the isomerase activity is performed later in the folding process.

Members of the a-carbonic anhydrase (α-CA) gene family encode not only proteins that exhibit the characteristic catalytic activity of CA (i.e., the reversible hydration of CO2), but also CA-related proteins that are apparently devoid of this activity. For a recent summary of the activity mechanisms and functions of the mammalian CA isozymes, see Sly and Hu (1995). In amniotes (reptiles, birds, and mammals), the active CA isozymes have been designated, CA I — CA VII, CA IX, CA XII, and CA XIII, and the presumed inactive isoforms, CA-RP VIII, CA-RP X, and CA-RP-XI. In non-amniotes, apparently inactive α-CA-related proteins (CAH-RPs) have been identified in both the nematode, C. elegans, and in the yam (Dioscorea). Additional CA-related proteins are present as transmembrane proteins in several pox viruses, and as the CA-RP N-terminal domains of the extracellular regions of the transmembrane proteins of receptor protein tyrosine phosphatases β and γ (RPTPβ and γ). All of these presumably “acatalytic” CA-related isoforms probably either have no, or greatly diminished, CO2 hydration activity due to the substitution of one or more of the three histidine residues that are required to bind the zinc ion that is essential for efficient CA activity.

Carbonic anhydrase VII (CA VII) appears to be the most highly conserved of the active mammalian carbonic anhydrases. We have characterized the catalytic activity and inhibition properties of a recombinant murine CA VII. CA VII has steady-state constants similar to two of the most active isozymes of carbonic anhydrase, CA II and IV; also, it is very strongly inhibited by the sulfonamides ethoxzolamide and acetazolamide, yielding the lowest Ki values measured by the exchange of 18O between CO2 and water for any of the mammalian isozymes of carbonic anhydrase. The catalytic measurements of the hydration of CO2 and the dehydration of HCO3- were made by stopped-flow spectrophotometry and the exchange of 18O using mass spectrometry. Unlike the other isozymes of this class of CA, for which Kcat/K(m) is described by the single ionization of zinc-bound water, CA VII exhibits a pH profile for Kcat/K(m) for CO2 hydration described by two ionizations at pKa 6.2 and 7.5, with a maximum approaching 8 x 10(7) M-1 s-1. The pH dependence of kcat/K(m) for the hydrolysis of 4-nitrophenyl acetate could also be described by these two ionizations, yielding a maximum of 71 M-1 s-1 at pH > 9. Using a novel method that compares rates of 18O exchange and dehydration of HCO3-, we assigned values for the apparent pKa at 6.2 to the zinc-bound water and the pKa of 7.5 to His 64. The magnitude of Kcat, its pH profile, 18O-exchange data for both wild-type and a H64A mutant, and inhibition by CuSO4 and acrolein suggest that the histidine at position 64 is functioning as a proton-transfer group and is responsible for one of the observed ionizations. A truncation mutant of CA VII, in which 23 residues from the amino-terminal end were deleted, has its rate constant for intramolecular proton transfer decreased by an order of magnitude with no change in Kcat/K(m). This suggests a role for the amino-terminal end in enhancing proton transfer in catalysis by carbonic anhydrase.

The alpha-carbonic anhydrase (alpha-CA) gene family in mammals encodes 10 CA or CA-like proteins (CA I-CA X). Although the gene for human CA VII has been cloned and characterized, the corresponding protein has not previously been purified, and hence, the CO2 hydrase activity of its product has not as yet been demonstrated. In this study, we have cloned the mouse CA VII cDNA in an E. coli, glutathione-S-transferase (GST) expression vector. The CO2 hydrase activity of the expressed protein is about 4% that of the high-activity CAII isozyme, demonstrating that this evolutionarily highly conserved protein is a catalytically active member of this CA gene family.

The unique structure of a protein is encoded in its characteristic sequence of amino acids; the processes by which this linear sequence collapses into a unique 3D structure remains an unsolved problem that represents one of the most challenging issues in fundamental biomolecular science. This so-called protein folding problem is the second half of the genetic code. Studies of this biological problem are complicated by the need to study dynamic behavior involving small populations of transient species in a solution environment. However, the use of advanced transient laser spectroscopy techniques based on intrinsic chromophores provides a powerful means to study this problem. Specifically, time-resolved phosphorescence of tryptophan (Trp) provides a means to study the dynamics associated with different regions of the protein surrounding the emitting Trp residue. Using these methodologies, we are able to study, in real time, the later stages of unfolding and refolding of the bacterial protein alkaline phosphatase, a nonspecific monoesterase. Results show the presence of several intermediate states, including states with significantly altered core structure that still exhibit complete biological activity. Moreover, the refolding of alkaline phosphatase following denaturation in either chaotropic denaturants or low pH reveals a relatively fast refolding leading to the biologically active state, while laser spectroscopy measurements show a soft core which is annealing to the native-like state on a time-scale long compared to the return of activity. The active refolded protein is also initially characterized by an increase in susceptibility to denaturant. The slow annealing of the core is consistent with the presence of high energy barriers that separate fully active, long-lived, kinetic intermediate states along the folding pathway, a description suggested in the rugged energy landscape model.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

When Escherichia coli alkaline phosphatase (AP) is refolded in vitro after extensive denaturation in 6.2 M guanidine hydrochloride, the enzymatic activity reaches its asymptotic value in 1 h at 24 degrees C. In contrast, the structural rigidity of the hydrophobic core of the protein, monitored by the recovery of the tryptophan phosphorescence lifetime, returns to its characteristic native-like value over several days. Moreover, the protein lability, measured by the rate of inactivation in 4.5 M guanidine hydrochloride, also changes on a time scale much longer than the recovery of activity. These results clearly demonstrate that while the return of enzymatic activity, the traditional measure of the attainment of the native state, indicates that AP has refolded to its final, active conformation, the phosphorescence data indicate otherwise. In the context of the rugged energy landscape model [Frauenfelder, H., et al. (1991) Science 254, 1598-1603], the slow annealing of the hydrophobic core is consistent with the presence of high-energy barriers that separate fully active intermediates along the folding pathway. The data suggest that the core of the protein undergoes continued structural rearrangements affecting the rigidity of the protein environment surrounding the emitting tryptophan and the protein lability long after the return of enzyme activity.

The room temperature phosphorescence of native human carbonic anhydrase (CA), and several mutants of this enzyme has been investigated. In these mutants the seven tryptophan residues in the native protein have sequentially been replaced by cysteine or phenylalanine. All of the mutants as well as native CA show room-temperature tryptophan phosphorescence (RTP) spectra. Surprisingly, only small differences in RTP life-times are noticeable among these mutants, indicating that there is more than one tryptophan residue with similar phosphorescence decay kinetics in the protein. The present results illustrate the danger in attributing the room temperature phosphorescence of a multi-tryptophan protein to a particular residue based solely on an analysis of the protein structure.

A variant allele at the CA I locus that produces a deficiency of erythrocyte-specific CA I occurs as a widespread polymorphism in pigtail macaques from southeast Asia. Sequence analyses revealed a C----G substitution 12 nucleotides downstream of the cap site in the variant erythrocyte CA I mRNA. This mutation forms a new AUG start site and an open reading frame coding for 26 amino acids that terminates 6 nucleotides before the normal AUG initiation codon for CA I. It appears that the presence of this upstream open reading frame greatly diminishes reinitiation of translation from the normal start site, resulting in trace levels of CA I in erythrocytes. Preferential use of the first AUG codon supports the scanning model for translation initiation in eukaryotes.

Two, electrophoretically distinct, forms of carbonic anhydrase I (CA Ia and CA Ib) are found at high polymorphic frequencies in red cells of natural populations of pigtail macaques, Macaca nemestrina, from southeast Asia. By use of the polymerase chain reaction, exons of the CA I gene were amplified from homozygous (a/a, b/b) and heterozygous (a/b) animals. Direct sequencing of the amplified DNA from four animals revealed differences between the a and the b electrophoretic alleles ranging from three to six nucleotides, and from one to three differences within each allele. These results indicate a greater genetic variability at the CA I locus in this macaque species than previously realized.

There are several evolutionarily invariant amino acids in the primary structures of all known isoenzymes of carbonic anhydrase. One of these is Ser-29 which is situated in the peripheral part of the active site interacting by hydrogen bonds with amino acids located nearby in the tertiary structure. Furthermore, the neighbourhood of Ser-29, composed of Gln-28, Pro-30, Tyr-194, Ser-197 and Trp-209, has a totally invariant structure. The structural role of Ser-29 was investigated by site-directed mutagenesis. The stability of two enzyme mutants, where Ser-29 was replaced by alanine and cysteine, towards denaturation by guanidine-HCl was studied. Changing Ser-29 to Ala resulted in a destabilization by 2.6 kcal/mol, corresponding to the loss of 2-3 hydrogen bonds. Interestingly, Ser-29 is within hydrogen bond distance to Tyr-194, Ser-197 and Trp-209 in the tertiary structure. Therefore, rupture of these interactions caused by the Ser-29----Ala substitution could explain the observed destabilization of this enzyme variant. Substituting cysteine for Ser-29 gives rise to a drastic decrease in the stability of the protein (change in midpoint concentration of denaturation from 0.96 M to less than 0.1 M guanidine-HCl) despite the minor structural change (O----S atom). This destabilization corresponds to approx. 7-8 kcal/mol and cannot be explained by changes in hydrogen bond pattern only, but must also include unfavourable conformational changes to avoid van der Waals collisions originating from the somewhat larger thiol group.

Bovine muscle carbonic anhydrase III was purified to homogeneity by the strategy of rapid ion-exchange chromatography. The ionic exchanger used was CM-cellulose, and this is the first application of this technique on a cation exchanger. Nitrogen gas was used to pressurize the chromatographic column to accelerate the elution. The results show that proteins with high isoelectric points can also be purified in this way. The procedure is very time-saving compared with conventional chromatography, reducing the elution time five- to ten-fold. The proteins are in addition protected against oxidation by air.

1. 1. A partial primary structure (197 residues) of carbonic anhydrase from tiger shark ( Galeocerdo cuvieri ) erythrocytes has been determined. 2. 2. The amino acid sequence of the enzyme is identical to those of human cytoplasmic carbonic anhydrases (CA I-III) by as much as 52–60%. 3. 3. It is shown that tiger shark CA most closely resembles the CA II isoenzyme of amniotes. 4. 4. Isoelectric focusing and inhibition studies on carbonic anhydrase from dogfish ( Squalus acanthias ) blood and muscle indicate the presence of the same isoenzyme in shark blood and muscle.

A method of rapid ion-exchange chromatography of DEAE-cellulose for preparative purposes is described. Basically, the flow-rate is increased by applying an air pressure on the column. By this technique it is possible to purify gram quantities of protein in 2–4 h with acceptable resolution. In preparations of bovine and porcine carbonic anhydrases the elution times were reduced by a factor of about ten compared to those of conventional methods. The enzymes purified in this way showed a high degree of homogeneity. The method should be generally applicable in protein purification, and especially advantageous in purification of unstable proteins where time-consuming separations often give rise to low yields of active material.

In this study it is shown that the higher molecular weight prevously reported for tiger shark carbonic anhydrase (carbonate hydro-layse, EC 4.2.1.1) compared to other carbonic anhydrases is decreased to a normal value around 30 000 after disulfide reduction of the enzyme. This difference in molecular weight is at least partly due to the existence of disulfide-linked glutathione and cysteine residues. Approx. 3 mol glutathione and a similar amount of cysteine are shown to be bound per mol enzyme. The presence of these factors also has effects on the enzyme activity.

Kinetic studies of the folding of carbonic anhydrase have indicated the occurrence of various conformational intermediates. Human carbonic anhydrase I contains a single cysteine residue, Cys-212, which in the native state is unavailable for alkylation. In the unfolded state, it can be specifically modified with iodoacetate. In this study the accessibility of Cys-212 in human carbonic anhydrase I to iodo[2-14C]acetate during the refolding process has been investigated. It is shown that Cys-212 is hidden to the alkylating agent as soon as the refolding is initiated. Since Cys-212 is located in the extensive beta-structure passing through the enzyme, it appears that the Cys-containing beta-strand is part of a rapidly formed nucleation center created during the folding process. This beta-strand (No. 7) together with its neighboring beta-strand (No. 6) constitute the most hydrophobic regions of the enzyme. Because hydrophobic contacts are considered to be important in predicting nucleation sites, these beta-strands probably partake in the formation of the nucleation center. These beta-strands are also partly involved in the bottom region of the active site cavity, indicating that this region is formed during the initial folding events. As a result of this study it was also observed that 2-mercaptoethanol is a potent inhibitor of the enzyme with a K1 = 26 microM at pH 8.0.

A modification of affinity electrophoresis for preparative purposes is described. This method has been applied to the purification of human erythrocyte carbonic anhydrases B and C. During conventional affinity chromatography some hemoglobin contamination occurs. By introduction of an electrophoretic purification step after the immobilization of carbonic anhydrase to the affinity gel, the hemoglobin impurity is reduced about eight and two times in the preparations of the B and C enzymes, respectively, compared to the enzymes purified by affinity chromatography.

Conditions for reactivation of guanidine-HCl-denatured bovine Co(II)-carbonic anhydrase II are given. The renaturation is accompanied by recovery of the native Co(II)-spectrum of the enzyme. After studying the kinetics of the renaturation process, the metal ion involvement in the refolding pathway can be summarized as follows: (1) Formation of an inactive Co(II)-intermediate with the metal ion firmly bound. No native Co(II)-spectrum is observed in this state, probably due to octahedral coordination of the metal ion in this intermediate. (2) Formation of an inactive Co(II)-intermediate with a native Co(II)-spectrum. The final tetrahedral coordination of the metal ion seems to have been formed in this state. (3) Formation of the active conformation of the enzyme. A functioning active-site is formed after some rearrangements of the polypeptide chain. This isomerisation step does not need to be preceded by formation of the intermediate with a native Co(II)-spectrum. Coordination of Co2+ in a native-like manner is, however, a prerequisite for enzymic activity. It is tentatively suggested that the metal ion is involved in stabilizing a nucleation structure formed at the bottom of the active centre. This probably occurs through binding of Co2+ to some or all of its histidyl ligands in this region after an early structuration of the metal ion binding site. The binding mechanisms of Co2+ appear to be similar for the refolding enzyme and the native apoenzyme, inferring that the binding site formed as a result of the nucleation process probably has the same structure as in the native conformation.