Lipoprotein lipase isoelectric point isoforms in humans

Lipoprotein lipase (LPL) hydrolyzes circulating triacylglycerols (TAG) into free fatty acids and glycerol. It is present in almost all tissues and its tissue-specific regulation directs the flow of circulating TAG in the body. We demonstrated in a previous study that, in rat heart and post-heparin plasma (PHP), LPL consists of a pattern of more than 8 forms of the same apparent molecular weight, but different isoelectric point (pI). In the present study we describe, for the first time, the existence of at least nine LPL pI isoforms in human PHP, with apparent pI between 6.8 and 8.6. Separation and characterization of these forms was carried out by 2DE combined with Western blotting and mass spectrometry (MALDI-TOF/MS and LC–MS/MS). Further studies are needed to discover their molecular origin, the pattern of pI isoforms in human tissues, their possible physiological functions and possible modifications of their pattern in different pathologies.     

A novel rat homolog of the Saccharomyces cerevisiae ubiquitin-conjugating enzymes UBC4 and UBC5 with distinct biochemical features is induced during spermatogenesis.

The Saccharomyces cerevisiae ubiquitin-conjugating enzymes (E2s) UBC4 and UBC5 are essential for degradation of short-lived and abnormal proteins. We previously identified rat cDNAs encoding two E2s with strong sequence similarity to UBC4 and UBC5. These E2 isoforms are widely expressed in rat tissues, consistent with a fundamental cellular function for these E2s. We now report a new isoform, 8A, which despite having >91% amino acid identity with the other isoforms, shows several novel features. Expression of the 8A isoform appears restricted to the testis, is absent in early life, but is induced during puberty. Hypophysectomy reduced expression of the 8A isoform. In situ hybridization studies indicated that 8A mRNA is expressed mainly in round spermatids. Immunoblot analyses showed that 8A protein is found not only in subfractions of germ cells enriched in round spermatids but also in subfractions containing residual bodies extruded from more mature elongated spermatids, indicating that the protein possesses a longer half-life than the mRNA. Unlike all previously identified mammalian and plant homologs of S. cerevisiae UBC4, which possess a basic pI, the 8A isoform is unique in possessing an acidic pI. The small differences in sequence between the 8A isoform and other rat isoforms conferred differences in biochemical function. The 8A isoform was less effective than an isoform with a basic pI or ineffective in conjugating ubiquitin to certain fractions of testis proteins. Thus, although multiple isoforms of a specific E2 may exist to ensure performance of a critical cellular function, our data demonstrate, for the first time, that multiple genes also permit highly specialized regulation of expression of specific isoforms and that subtle differences in E2 primary structure can dictate conjugation of ubiquitin to different subsets of cellular proteins.     

Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity

A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic (lower pI) peaks were found to represent deamidated and sialyated species. Higher pI peaks were found to contain N- and C-terminal heavy-chain variants. Biological activities of the more abundant isoforms were found to be comparable. An approach streamlining the characterization of antibody charge heterogeneity is proposed.     

Hamster contraception associated protein 1 (CAP1)

Based on cDNA and amino acid sequence, we demonstrate that hamster contraception associated protein 1 (CAP1) protein (an homolog of DJ-1 in mouse, CAP1/SP22/RS in rat and DJ-1/RS in human) is conserved during evolution. Through solubilization studies, it was demonstrated that hamster CAP1 has a peripheral membrane localization. SDS-PAGE analysis revealed that the migration pattern for hamster CAP1 compared to the other rodent counterparts, rat and mouse was different; indicating species-specific differences in the protein (possibly due to post-translational modifications). This protein also shows a ubiquitous presence in both somatic and germ tissues, and has been localized to the sperm tail. It was noticed that hamster CAP1 was lost from the mid piece of spermatozoa during capacitation. Interestingly, following in vitro treatment with ornidazole, CAP1 was lost from the spermatozoa and immunofluorescence studies showed that the major loss was from the mid piece of the spermatozoa. Another interesting feature highlighted about hamster CAP1 is its tendency to exist in two pI isoforms. Summarily, hamster CAP1 appears to exhibit species-specific differences compared to its rodent counterparts with respect to its unique peripheral localization, its size, two pI isoforms, and fate during capacitation, which may have implications in its functions.     

Mapping and structural dissection of human 20 S proteasome using proteomic approaches

The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (alpha7beta7beta7alpha7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the alpha and beta subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pI values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the alpha7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser(250).     

Molecular forms of rat angiotensinogen in plasma and brain: identification by isoelectric focusing and immunoblot analysis

Angiotensinogen (Ao) is the glycoprotein precursor of the vasoactive peptide angiotensin II. While Ao is synthesized as multiple molecular forms, the biochemical characteristics of this protein in blood and other tissues have not been defined. In this study, the charge heterogeneity of Ao in rat plasma, cerebrospinal fluid and that secreted by astrocyte and neuronal cultures was examined using analytical isoelectric focusing in combination with immunoblotting and quantitative image analysis. Normal rat male plasma Ao separated into 9 isoforms in the pI range 4.34-4.92 (1, 4.34; 2, 4.41; 3, 4.48; 4, 4.58; 5, 4.61; 6, 4.66; 7, 4.68; 8, 4.81; 9, 4.92); the percentage contribution of each to total plasma Ao was 13, 20, 23, 18, 2, 7, 10, 5, and < 1, respectively. A similar isoelectric focusing pattern was observed in female rat plasma with the exception that the relative contribution of isoform 6 was reduced to 2% of total Ao. Cerebrospinal fluid Ao displayed a more diverse charge heterogeneity than plasma Ao, focusing over a broader pI range of 4.42-5.24. Astrocytes and neurons secreted Ao isoforms in the pI range 4.44-5.29 and 4.42-4.95, respectively, with the astrocyte cultures showing additional bands towards the cathode. It was concluded that rat Ao is secreted as multiple charged forms that are regulated in a sex- and cell-specific manner. These differences between plasma and brain Ao suggest a functional diversity, a view which is supported by recent evidence linking Ao variants to hypertension.     

Isolation and characterization of human glucose-6-phosphate isomerase isoforms containing two different size subunits

Previously undetected isoforms of human glucose-6-phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK.     

Analysis of photoaffinity-labeled aryl hydrocarbon receptor heterogeneity by two-dimensional gel electrophoresis

The level of charge heterogeneity in the aryl hydrocarbon receptor (AhR) was examined by high-resolution denaturing two-dimensional (2D) gel electrophoresis. Hepa 1c1c7 cell cytosolic fraction was photoaffinity-labeled with 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin and applied to isoelectric focusing (IEF) tube gels. After optimization of focusing conditions a broad peak of radioactivity was detected in the apparent pI range of 5.2-5.7. IEF tube gels were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by visualization of the radiolabeled AhR by autoradiography; three distinct isoforms were detected. The same 2D electrophoretic isoform pattern was obtained when the AhR from Hepa 1c1c7 was photoaffinity-labeled in cell culture. BPrCl cells, a mutant line derived from Hepa 1c1c7 cells, contain an AhR that is unable to bind to DNA. Photoaffinity-labeled BPrCl cytosolic fractions were subjected to 2D gel electrophoretic analysis resulting in essentially the same molecular weight and isoform pattern as seen in Hepa 1c1c7 cytosol. This result would suggest that if a mutation is present in the BPrCl AhR it has not caused a significant change in its IEF pattern, although a small shift in the pI values was observed. Two-dimensional gel electrophoresis of photoaffinity-labeled cytosolic fractions from HeLa cells, the rat liver tumor cell line McA-RH7777, and buffalo rat thymus revealed three isoforms, essentially the same isoform pattern as in Hepa 1c1c7 cells. This would indicate that despite the considerable molecular weight polymorphism between species the level of charge heterogeneity is highly conserved.     

Characterization of proliferating cell nuclear antigen (PCNA) isoforms in normal and cancer cells: there is no cancer-associated form of PCNA

In order to clarify the status of PCNA in normal and transformed cells, we performed analysis of this protein by 2D-PAGE, Western blot and mass spectrometry. All the cell lines examined contained the major PCNA form (pI 4.57/30kDa), that is not post-translationally modified. In addition to the major form, two minor isoforms (pI 4.52/30kDa and pI 4.62/30kDa) were also detected in all the cell lines examined. However, the level of PCNA in cancer cells is 5-6 folds higher than those in primary and most of the immortalized cells. Taken together, the significant difference in PCNA status between cancer and normal cells is not at the post-translational modifications but in the overall levels of PCNA.     

Two-dimensional electrophoresis of recombinant human erythropoietin: a future method for the European Pharmacopoeia?

Quality assurance of recombinant protein drugs concerning identity and purity represents a difficult task, in particular, when post-translational modifications lead to a heterogeneous mixture of biomolecules. We chose Neorecormon (rh-EPO, Roche) for our studies to demonstrate the efficiency of two-dimensional electrophoresis (2-DE) to analyse post-translationally modified recombinant drugs. More than 40 protein spots in the range from isoelectric point (pI) 3.5-4.5 and 32-45 kDa could be separated. Enzymatic deglycosylation revealed that the heterogeneity of the protein pattern is mainly caused by variations in glycosylation. In comparison to the separately performed isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as requested by the European Pharmacopoeia, we see a great synergy to use 2-DE for the analysis of rh-EPO. A by far higher resolution can be achieved, allowing an improved differentiation of the various rh-EPO glycoforms. Sequential deglycosylation of sialic acids, N-glycosides and the O-glycoside lead to significant shifts both in apparent relative molecular mass and pI. Comparing the 2-DE patterns of rh-EPO before and after deglycosylation allows on the one hand valuable information to be gained on the glycosylation of the recombinant protein and shows on the other hand how significantly the 2-DE protein pattern can be influenced by the glycosylation. As the equipment for the performance of 2-DE has improved significantly over the last decade, we see 2-DE as a reliable method, which should be approved for the routine quality assurance of recombinant drugs and also recommended for the European Pharmacopoeia.     

Cardiac fatty acid-binding proteins. Isolation and characterization of the mitochondrial fatty acid-binding protein and its structural relationship with the cytosolic isoforms

In the course of our studies on the structural diversity of the isoforms of cardiac fatty acid-binding proteins (cFABPs), a cardiac-type FABP from the matrix of bovine heart mitochondria was purified to homogeneity and obtained as a single 15-kDa protein with an isoelectric point of 4.9. The primary structures of this protein and of the two isoforms isolated from the cytosol (pI4.9-cFABP and pI 5.1-cFABP) were investigated by means of plasma desorption mass spectrometry and sequencing of peptides. All three proteins are amino-terminally blocked with an acetyl group and shown to be colinear with the sequence deduced from a cDNA clone for bovine heart fatty acid-binding protein (Billich, S., Wissel, T., Kratzin, H., Hahn, U., Hagenhoff, B., Lezius, A. G., and Spener, F. (1988) Eur. J. Biochem. 175, 549-556) except for the residue at position 98. This residue is demonstrated to be the molecular origin of bovine cFABP isoforms since pI 5.1-cFABP contains Asn98 in accordance with the sequence derived from the cDNA, whereas in pI 4.9-cFABP, this position is occupied by Asp98. Moreover, mitochondrial FABP is identical to pI 4.9-cFABP. Molecular masses of pI 4.9-cFABP (14,679 +/- 10 Da) and pI 5.1-cFABP (14,678 +/- 20 Da) determined by plasma desorption mass spectrometry coincide with that calculated from the cDNA (14,673 Da). Hence, residues linked to these proteins by posttranslational modification are not present, and the Asn-Asp exchange is the sole origin of heterogeneity of mitochondrial and cytosolic fatty acid-binding proteins from bovine heart.     

The multifunctional poly(A)-binding protein (PABP) 1 is subject to extensive dynamic post-translational modification, which molecular modelling suggests plays an important role in co-ordinating its activities

PABP1 [poly(A)-binding protein 1] is a central regulator of mRNA translation and stability and is required for miRNA (microRNA)-mediated regulation and nonsense-mediated decay. Numerous protein, as well as RNA, interactions underlie its multi-functional nature; however, it is unclear how its different activities are co-ordinated, since many partners interact via overlapping binding sites. In the present study, we show that human PABP1 is subject to elaborate post-translational modification, identifying 14 modifications located throughout the functional domains, all but one of which are conserved in mouse. Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations, and lysine acetylations. The latter dramatically alter the pI of PABP1, an effect also observed during the cell cycle, suggesting that different biological processes/stimuli can regulate its modification status, although PABP1 also probably exists in differentially modified subpopulations within cells. Two lysine residues were differentially acetylated or methylated, revealing that PABP1 may be the first example of a cytoplasmic protein utilizing a 'methylation/acetylation switch'. Modelling using available structures implicates these modifications in regulating interactions with individual PAM2 (PABP-interacting motif 2)-containing proteins, suggesting a direct link between PABP1 modification status and the formation of distinct mRNP (messenger ribonucleoprotein) complexes that regulate mRNA fate in the cytoplasm.     

Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes

Lipoprotein lipase (LPL) is the enzyme responsible for hydrolysis of circulating triglyceride-rich lipoproteins and is important for storage of adipocyte lipid. To study the regulation of LPL synthetic rate in adipose tissue, primary cultures of isolated rat adipocytes were pulse-labeled with [35S]methionine, and LPL was immunoprecipitated with an LPL-specific antibody. A pulse-chase experiment identified the cellular and secreted forms of LPL as a 55-57-kDa protein. In the presence of heparin, there was a large increase in secretion of newly synthesized LPL from the cells, although heparin did not stimulate cellular LPL synthetic rate. When cells were exposed to insulin for 2 h, pulse-labeling revealed that insulin stimulated a maximal dose-related increase in LPL synthetic rate of 300% of control. This increase in LPL synthetic rate was observed after an exposure to insulin for as little as 60 min and was accompanied by only a 10-25% increase in total protein synthesis. In addition, insulin had no effect on the turnover of intracellular LPL. Using a cDNA probe for LPL, insulin induced a 2-fold increase in the LPL mRNA. Thus, insulin stimulated an increase in specific LPL mRNA in isolated rat adipocytes. This increase in LPL mRNA then leads to an increase in the synthetic rate of the LPL protein.     

Heterogeneity of tyrosinases isolated from rat skin, normal human skin, and human melanoma tumors

An electrophoretically homogeneous preparation of tyrosinase (Mr = 61 kDa) was isolated from rat skin. The purification procedure which consisted in chromatographic separation of Triton X-100-solubilized proteins included four main steps, namely: gel filtration, anion-exchange chromatography and two consecutive affinity chromatography steps. Isoelectrofocusing revealed the presence of 9 isoforms possessing an L-DOPA oxidase activity, of which proteins with pI of 4.26 and 4.33 were the major ones. The specific activity of the preparation was 43 nmol/min/mg of protein. Human skin epidermis was practically devoid of the L-DOPA-oxidase activity which was due not only to the absence of tyrosinase but also to the presence of a large amount of a 66 kDa protein able to inhibit the oxidation of L-DOPA to DOPA-chrom. The tyrosinase preparation from human melanoma consisted, predominantly, of two isoforms (48 and 69 kDa) which upon isoelectrofocusing displayed a high heterogeneity at pH around 3-5. The specific activity of the melanoma preparation markedly exceeded that of normal skin tyrosinase and was equal to 290 nmol/min/mg of protein.     

Apolipoprotein E content of VLDL limits LPL-mediated triglyceride hydrolysis

High levels of circulating triglycerides (TGs), or hypertriglyceridemia, are key components of metabolic diseases, such as type 2 diabetes, metabolic syndrome, and CVD. As TGs are carried by lipoproteins in plasma, hypertriglyceridemia can result from overproduction or lack of clearance of TG-rich lipoproteins (TRLs) such as VLDLs. The primary driver of TRL clearance is TG hydrolysis mediated by LPL. LPL is regulated by numerous TRL protein components, including the cofactor apolipoprotein C-II, but it is not clear how their effects combine to impact TRL hydrolysis across individuals. Using a novel assay designed to mimic human plasma conditions in vitro, we tested the ability of VLDL from 15 normolipidemic donors to act as substrates for human LPL. We found a striking 10-fold difference in hydrolysis rates across individuals when the particles were compared on a protein or a TG basis. While VLDL TG contents moderately correlated with hydrolysis rate, we noticed substantial variations in non-apoB proteins within these particles by MS. The ability of LPL to hydrolyze VLDL TGs did not correlate with apolipoprotein C-II content, but it was strongly inversely correlated with apolipoprotein E (APOE) and, to a lesser extent, apolipoprotein A-II. Addition of exogenous APOE inhibited LPL lipolysis in a dose-dependent manner. The APOE3 and (particularly) APOE4 isoforms were effective at limiting LPL hydrolysis, whereas APOE2 was not. We conclude that APOE on VLDL modulates LPL activity and could be a relevant factor in the pathogenesis of metabolic disease.     

Mammalian proteasome subpopulations with distinct molecular compositions and proteolytic activities

The proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far have yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multiprotein complexes in a laminar flow that support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26 with additional subpopulations focusing in the range from pH 5.10 to 5.33. In contrast, the majority of hepatic 20 S proteasomes had a pI of 5.05 and focused from pH 5.01 to 5.29. Importantly proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities because proteasomes with pI 5.21 contained 58% less of the inducible subunit beta 2i compared with those with pI 5.28. In addition, dephosphorylation of 20 S proteasomes demonstrated that besides molecular composition posttranslational modifications largely contribute to their pI values. These data suggest the possibility of mixed 20 S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery. Thus, proteasome subpopulations may serve as novel therapeutic targets in the myocardium.     

Heterogeneity of serum gelatinases MMP‐2 and MMP‐9 isoforms and charge variants

The matrix metalloproteinases (MMPs) gelatinase A (MMP‐2) and gelatinase B (MMP‐9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2‐DZ) as an extension of classic monodimensional zymography (1‐DZ) to analyse the complete pattern of isoforms and post‐translational modifications of both MMP‐9 and MMP‐2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP‐2 isoforms, and at least three different isoforms and two different states of organization of MMP‐9 (the multimeric MMP‐9 and the N‐GAL‐MMP‐9 complex) were observed. In addition, 2‐DZ revealed for the first time that all MMP‐9 and MMP‐2 isoforms actually exist in the form of charge variants: four or five variants in the N‐GAL complex, more charge variants in the case of MMP‐9; and five to seven charge variants for MMP‐2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP‐9) and phosphorylation (MMP‐2) contributed to molecular heterogeneity. The detection of charge variants of MMP‐9 and MMP‐2 in MS serum samples illustrates the power of 2‐DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases.     

Differential recruitment of PKC isoforms in HeLa cells during redox stress

The protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation.