Distinct Cytoplasmic Granules Research Articles

Membrane damage by MBP-1 is mediated by pore formation and amplified by mtDNA

Eosinophils play a crucial role in host defense while also contributing to immunopathology through the release of inflammatory mediators. Characterized by distinctive cytoplasmic granules, eosinophils securely store and rapidly release various proteins exhibiting high toxicity upon extracellular release. Among these, major basic protein 1 (MBP-1) emerges as an important mediator in eosinophil function against pathogens and in eosinophil-associated diseases. While MBP-1 targets both microorganisms and host cells, its precise mechanism remains elusive. We demonstrate that formation of small pores by MBP-1 in lipid bilayers induces membrane permeabilization and disrupts potassium balance. Additionally, we reveal that mitochondrial DNA (mtDNA) present in eosinophil extracellular traps (EETs) amplifies MBP-1 toxic effects, underscoring the pivotal role of mtDNA in EETs. Furthermore, we present evidence indicating that absence of CpG methylation in mtDNA contributes to the regulation of MBP-1-mediated toxicity. Taken together, our data suggest that the mtDNA scaffold within extracellular traps promotes MBP-1 toxicity.

Expression of Antimicrobic Peptide Piscidin1 in Gills Mast Cells of Giant Mudskipper Periophthalmodon schlosseri (Pallas, 1770).

The amphibious teleost Giant mudskipper (Periophthalmodon schlosseri, Pallas 1770) inhabit muddy plains and Asian mangrove forests. It spends more than 90% of its life outside of the water, using its skin, gills, and buccal-pharyngeal cavity mucosa to breathe in oxygen from the surrounding air. All vertebrates have been found to have mast cells (MCs), which are part of the innate immune system. These cells are mostly found in the mucous membranes of the organs that come in contact with the outside environment. According to their morphology, MCs have distinctive cytoplasmic granules that are released during the degranulation process. Additionally, these cells have antimicrobial peptides (AMPs) that fight a variety of infections. Piscidins, hepcidins, defensins, cathelicidins, and histonic peptides are examples of fish AMPs. Confocal microscopy was used in this study to assess Piscidin1 expression in Giant Mudskipper branchial MCs. Our results demonstrated the presence of MCs in the gills is highly positive for Piscidin1. Additionally, colocalized MCs labeled with TLR2/5-HT and Piscidin1/5-HT supported our data. The expression of Piscidin1 in giant mudskipper MCs highlights the involvement of this peptide in the orchestration of teleost immunity, advancing the knowledge of the defense system of this fish.

Observations on the cytomorphology and ultrastructure of the peripheral blood cells of native cattle (Zobawng) of Mizoram, India.

This study aims to characterize the cytomorphology and ultrastructure of blood cells of native cattle of Mizoram. Twelve numbers of blood samples (10ml) were collected from the Zobawng cattle, irrespective of sex. Blood smears were prepared and stained with different stains for cytomorphological study. The standard protocol has been followed for preparing blood samples for electron microscopy. Under a light microscope, erythrocytes of cattle were non-nucleated and round. The neutrophils were round, and the cytoplasm contained cytoplasmic granules. The eosinophils were rounded in outline with distinct cytoplasmic granules. The presence of basophils was infrequent with distinct blue color cytoplasmic granules. Small, medium, and large types of lymphocytes were recorded. The monocytes were round to oval in outline. Platelets were irregular to round. The reticulocytes were recorded occasionally, like small blue thin rods or granules. The cytoplasm and nucleus of granulocytes fluoresced greenish-yellow and red, respectively, with supravital stain. Under scanning electron microscopy (SEM), the erythrocytes appeared as biconcave discs. Different leukocytes were observed with their finger-like, plate-like, and narrow cell processes on their surface. Platelets were irregular structures. In transmission electron microscopy (TEM), the erythrocytes appeared anucleated biconcave elongated, neutrophils were roughly rounded with small cytoplasmic processes, and eosinophils were roughly circular with small cytoplasmic processes, the basophils were roughly circular with oval to elongated cytoplasmic granules, lymphocytes were roughly circular with centrally placed well-marked oval indented nucleus and some cytoplasmic processes, monocytes appeared spherical with long thick cytoplasmic processes and the non-nucleated platelets appeared roughly round to elongated. RESEARCH HIGHLIGHTS: Blood tests are a very good tool for determining animal health and diagnosing hematological illnesses. This study showed the cytomorphological and ultrastructural features of the blood cells of native cattle (Zobawng) of Mizoram state of India. The cytomorphological studies revealed that the morphological features of the blood cells of Zobawng cattle resembled to other domestic and wild animals, however, there were some differences in the cellular components. Nuclear DNA appeared orthochromatically greenish-yellow, but cytoplasmic RNA was observed to be metachromatically red under the fluorescence microscope. scanning electron microscopic (SEM) and transmission electron microscopic (TEM) results revealed that the ultrastructural features of the blood cells of Zobawng cattle contain certain cellular morphological differences as compared to the other domestic and wild animals.

Dual Functional Roles of Molecular Beacon as a MicroRNA Detector and Inhibitor

MicroRNAs are essential in many cellular processes. The ability to detect microRNAs is important for understanding its function and biogenesis. This study is aimed at using a molecular beacon to detect miR-430 in developing zebrafish embryos as a proof of principle. miR-430 is crucial for the clearance of maternal mRNA during maternal zygotic transition in embryonic development. Despite its known function, the temporal and spatial expression of miR-430 remains unclear. We used various imaging techniques, including laser scanning confocal microscopy, spinning disk, and lightsheet microscopy, to study the localization of miR-430 and any developmental defects possibly caused by the molecular beacon. Our results show that miR-430 is expressed early in development and is localized in distinct cytoplasmic granules where its target mRNA can be detected. We also show that the designed molecular beacon can inhibit the function of miR-430 and cause developmental defect in the brain, notochord, heart, and kidney, depending on the delivery site within the embryo, suggesting that miR-430 plays a diverse role in embryonic morphogenesis. When compared with morpholino, molecular beacon is 2 orders of magnitude more potent in inhibiting miR-430. Thus, our results reveal that in addition to being used as a valuable tool for the detection of microRNAs in vivo, molecular beacons can also be employed to inhibit microRNAs in a specific manner.

Sequestration-Mediated Downregulation of de Novo Purine Biosynthesis by AMPK.

Dynamic partitioning of de novo purine biosynthetic enzymes into multienzyme compartments, purinosomes, has been associated with increased flux of de novo purine biosynthesis in human cells. However, we do not know of a mechanism by which de novo purine biosynthesis would be downregulated in cells. We have investigated the functional role of AMP-activated protein kinase (AMPK) in the regulation of de novo purine biosynthesis because of its regulatory action on lipid and carbohydrate biosynthetic pathways. Using pharmacological AMPK activators, we have monitored subcellular localizations of six pathway enzymes tagged with green fluorescent proteins under time-lapse fluorescence single-cell microscopy. We revealed that only one out of six pathway enzymes, formylglycinamidine ribonucleotide synthase (FGAMS), formed spatially distinct cytoplasmic granules after treatment with AMPK activators, indicating the formation of single-enzyme self-assemblies. In addition, subsequent biophysical studies using fluorescence recovery after photobleaching showed that the diffusion kinetics of FGAMS were slower when it localized inside the self-assemblies than within the purinosomes. Importantly, high-performance liquid chromatographic studies revealed that the formation of AMPK-promoted FGAMS self-assembly caused the reduction of purine metabolites in HeLa cells, indicating the downregulation of de novo purine biosynthesis. Collectively, we demonstrate here that the spatial sequestration of FGAMS by AMPK is a mechanism by which de novo purine biosynthesis is downregulated in human cells.

Season-Induced Changes in the Liver Cells and Ovarian Tissues During Growth, Maturation, Spawning and Post-spawning Phases in Spiny Eel Mastacembelus armatus (Lacepède, 1800)

Cytological status of hepatocytes in respect to seasons influencing ovarian activities has been studied in Mastacembelus armatus (Hamilton). Histologically, the hepatocyte of female liver was provided with distinct nucleus and basophilic cytoplasmic granules. In M. armatus different germ line cells were recognized on the basis of size and histoarchitecture of the cells. By examining seasonal changes in relation to hepatocytes and ovary, it was found that during growth and maturation phases the density of cytoplasmic granules of hepatocytes was increased in number as well as the nucleus become hypertrophied. These features were well correlated with the occurrence of cortical alveolus and yolk granule stage in the ovary. During spawning phase the cytoplasmic granules were sparse in the hypertrophied hepatic cells. This was due to the dynamic cytological activities like vitellogenesis and occurrence of mature oocytes in the ovary. However, no significant alterations were noticed in the hepatocytes during post spawning phase due to the appearance of new germ line cells in the ovary. Cytological changes in the hepatocytes were correlated with the reproductive phases in female M. armatus.

Protein kinases are associated with multiple, distinct cytoplasmic granules in quiescent yeast cells.

The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival.

Methods for the study of mast cell recruitment and accumulation in different tissues.

Mast cells (MC) are important effector cells involved in a wide range of inflammatory diseases. The lineage-committed, tissue-localized progenitor (MCp) is not easily identified histochemically like the mature MC because they lack the distinctive cytoplasmic granules. However, they can be identified by their unique cell surface phenotype and by their ability to be expanded in culture using selective growth factors. Here we describe the methods that allow evaluation of MCp and mature MC in peripheral tissues under basal and inflammatory conditions. Thus, one can enumerate mature MC as well as immature committed progenitors in order to study basal homing, inflammatory recruitment, maturation, and life span. We also provide an analysis of difficulties that could emerge during these procedures.

Basophils are elevated in nasal polyps of patients with chronic rhinosinusitis without aspirin sensitivity

Basophils are elevated in nasal polyps of patients with chronic rhinosinusitis without aspirin sensitivity

Gastrointestinal Neuromuscular Pathology in Alpers Disease

Alpers disease is a recessive mitochondrial disorder caused by mutations in POLG1 and characterized primarily by progressive neurological and hepatic degeneration. Intestinal dysmotility is a frequent symptom, but it is often overshadowed by other clinical manifestations. The onset and progression of Alpers disease vary; however, most patients die during childhood, often before a specific diagnosis has been established. The gastrointestinal neuromuscular pathology of 4 patients, obtained largely from postmortem specimens, showed distinctive eosinophilic cytoplasmic granules in a subset of enteric ganglia and patchy atrophy of small intestinal muscularis externa. The cytoplasmic inclusions corresponded to abnormal mitochondria, which have been reported previously in another mitochondrial disorder (mitochondrial neurogastrointestinal encephalomyopathy) but not in Alpers disease. Recognition of these distinctive light microscopic findings, in an appropriate clinical setting, should prompt the evaluation of an underlying primary mitochondriopathy.

A population of tRNA-derived small RNAs is actively produced in Trypanosoma cruzi and recruited to specific cytoplasmic granules

Over the last years an expanding family of small RNAs (i.e. microRNAs, siRNAs and piRNAs) was recognized as key players in diverse forms of gene silencing and chromatin organization. Effectors functions of these small RNAs are achieved through ribonucleoprotein (RNP) complexes containing at their center an Argonaute/Piwi protein. Although these proteins and their small RNA-associated machinery can be traced back to the common ancestor of eukaryotes, this machinery seems to be entirely lost or extensively simplified in some unicellular organisms including Trypanosoma cruzi, which are unable to trigger RNAi related phenomena. Speculating about the presence of alternate small RNA-mediated pathways in these organisms, we constructed and analyzed a size-fractionated cDNA library (20–35 nt) from epimastigotes forms of T. cruzi. Our results showed the production of an abundant class of tRNA-derived small RNAs preferentially restricted to specific isoacceptors and whose production was more accentuated under nutritional stress. These small tRNAs derived preferentially from the 5′ halves of mature tRNAs and were recruited to distinctive cytoplasmic granules. Our data favor the idea that tRNA cleavage is unlikely to be the consequence of non-specific degradation but a controlled process, whose biological significance remains to be elucidated.

Disseminated Cutaneous Mast Cell Tumors with Epitheliotropism and Systemic Mastocytosis in a Domestic Cat

A 15-year-old female domestic, medium-haired cat presented to the referring veterinarian with a 2-month history of multiple, raised, disseminated, nodular skin lesions. A biopsy of 1 of the lesions was submitted to the Oklahoma Animal Disease Diagnostic Laboratory for evaluation. Histologically, there were multiple dermal nodules composed of sheets of neoplastic round cells. Multifocally, the neoplastic cells formed multiple small clusters of 3 to 5 cells within the epidermis. Distinct cytoplasmic granules were evident within the neoplastic cells with toluidine blue and Giemsa stains. The neoplastic cells were immunoreactive for c-KIT and lacked immunoreactivity for cluster of differentiation 3 with immunohistochemistry. Based on these findings, multiple epitheliotropic cutaneous mast cell tumors were diagnosed. The cat's health declined rapidly despite aggressive treatment, and the animal was humanely euthanatized. A complete necropsy revealed sheets of similar neoplastic mast cells within the spleen, liver, and individual cells scattered within the bone marrow. Exon 11 of the c-KIT messenger RNA from 1 of the cutaneous masses and the spleen was amplified with reverse transcription polymerase chain reaction, sequenced, and compared with the published c-KIT messenger RNA sequence from fetal cat tissues. The maximum identity was 100% for both tissue samples. To the authors' knowledge, the present report is the first to describe disseminated cutaneous mast cell tumors with epitheliotropism and systemic mastocytosis in a domestic cat.

Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation

In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs. In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo. Localization of P granules and their physical nature remain poorly understood. Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension. As with other liquids, P granules rapidly dissolved and condensed. Localization occurred by a biased increase in P granule condensation at the posterior. This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell. Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm.

Granular acute lymphoblastic leukemia: A case report and literature review

Authors report a rare case of granular acute lymphoblastic leukemia (ALL) in a 45-year-old woman with a history of multiple myeloma. The patient's lymphoblasts contained large numbers of distinctive cytoplasmic granules closely mimicking heavily granulated myeloblasts in acute myeloid leukemia. These blasts were completely negative for myeloperoxidase but positive for acid phosphatase and Periodic Acid-Schiff reaction by cytochemical staining. Immunophenotype analysis by immunohistochemistry clearly demonstrated precursor B-cell phenotype. Granular ALL occurs approximately 2 to 7% in childhood populations but is extremely rare in adults. Such cases may cause problematic distinction from myeloid differentiation and lead to misdiagnosis of acute myeloid leukemia. Ten cases of adult granular acute lymphoblastic leukemia described to date in the literature were also reviewed.

Combinatorial SNARE Complexes Modulate the Secretion of Cytoplasmic Granules in Human Neutrophils

Mobilization of human neutrophil granules is critical for the innate immune response against infection and for the outburst of inflammation. Human neutrophil-specific and tertiary granules are readily exocytosed upon cell activation, whereas azurophilic granules are mainly mobilized to the phagosome. These cytoplasmic granules appear to be under differential secretory control. In this study, we show that combinatorial soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with vesicle-associated membrane proteins (VAMPs), 23-kDa synaptosome-associated protein (SNAP-23), and syntaxin 4 underlie the differential mobilization of granules in human neutrophils. Specific and tertiary granules contained VAMP-1, VAMP-2, and SNAP-23, whereas the azurophilic granule membranes were enriched in VAMP-1 and VAMP-7. Ultrastructural, coimmunoprecipitation, and functional assays showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediated the rapid exocytosis of specific/tertiary granules, whereas VAMP-1 and VAMP-7 mainly regulated the secretion of azurophilic granules. Plasma membrane syntaxin 4 acted as a general target SNARE for the secretion of the distinct granule populations. These data indicate that at least two SNARE complexes, made up of syntaxin 4/SNAP-23/VAMP-1 and syntaxin 4/SNAP-23/VAMP-2, are involved in the exocytosis of specific and tertiary granules, whereas interactions between syntaxin 4 and VAMP-1/VAMP-7 are involved in the exocytosis of azurophilic granules. Our data indicate that quantitative and qualitative differences in SNARE complex formation lead to the differential mobilization of the distinct cytoplasmic granules in human neutrophils, and a higher capability to form diverse SNARE complexes renders specific/tertiary granules prone to exocytosis.

Regulation of the PI3K pathway by the eIF2α kinase PKR

Objective Study the role of PKR in PI3K signaling pathway. Two important steps of translation initiation include the recognition of the mRNA cap structure by eIF4E and the recycling of eIF2. Each step is thought to be regulated independently through the interaction of eIF4E with 4E binding proteins (4E-BPs) and the phosphorylation of the α subunit of eIF2 at serine 51. Results we demonstrate that the eIF2α kinase PKR provides a link between the two steps. PKR induces phosphoinositide-3 kinase (PI3K) activity leading to activation of Akt and the mammalian target of Rapamycin (mTOR) and phosphorylation of 4E-BP1. Despite 4E-BP1 phosphorylation, its interaction with eIF4E is enhanced in cells with activated PKR and occurs in distinct cytoplasmic granules containing phosphorylated eIF2α. Induction of the PI3K pathway antagonizes the apoptotic effects of PKR caused by eIF2α phosphorylation. PKR is also involved in the activation of PI3K and 4E-BP1 phosphorylation by serum or interferon (IFN)- 1 _. Conclusion Our data demonstrate a novel signaling property of PKR through the regulation of the PI3K pathway. Source of Funding This work has been supported by a grant from the Canadian Institutes of Health Research (CIHR) to Dr. A. Koromilas. S. Kazemi and D. Baltzis are both Research Students of the Terry Fox Foundation through awards from the National Cancer Institute of Canada (NCIC).

Expression of catalase mRNA and protein in adult rat brain: detection by nonradioactive in situ hybridization with signal amplification by catalyzed reporter deposition (ISH-CARD) and immunohistochemistry (IHC)/immunofluorescence (IF).

Catalase, the classical peroxisomal marker enzyme, decomposes hydrogen peroxide and is involved in the antioxidant defense mechanisms of mammalian cells. In addition, catalase can oxidize, by means of its peroxidatic activity, a variety of substrates such as methanol and ethanol, producing the corresponding aldehydes. The involvement of brain catalase in the oxidation of ethanol is well established, and severe afflictions of the CNS in hereditary peroxisomal diseases (e.g., Zellweger syndrome) are well known. Whereas the distribution of catalase in the CNS has been investigated by enzyme histochemistry and immunohistochemistry (IHC), very little is known about the exact localization of catalase mRNA in brain. Here we report the application of a tyramine/CARD (catalyzed reporter deposition)-enhanced nonradioactive in situ hybridization (ISH) protocol for detection of catalase mRNA in sections of perfusion-fixed, paraffin-embedded rat brain. Catalase mRNA could be demonstrated in a large number of neurons throughout the rat brain as a distinct cytoplasmic staining signal with excellent morphological resolution. Compared to our standard ISH protocol, the CARD-enhanced protocol for catalase mRNA detection in rat brain showed higher sensitivity and significantly better signal-to-noise ratio. In parallel IHC experiments, using an antigen retrieval method consisting of combined trypsin digestion and microwave treatment of paraffin sections, the catalase antigen was found as distinct cytoplasmic granules in most catalase mRNA-positive neurons. In addition, catalase-positive granules, presumably peroxisomes, were found by confocal laser scanning microscopy in glial cells, which were identified by double labeling immunofluorescence for GFAP and CNPase for astroglial cells and oligodentrocytes, respectively. The excellent preservation of morphology and sensitive detection of both mRNA and protein in our preparations warrant the application of the protocols described here for systematic studies of catalase and other peroxisomal proteins in diverse pathological conditions such as Alzheimer's disease and aging.

Cosmopolitan distribution of the large composite microbial mat spirochete, Spirosymplokos deltaeiberi.

Inocula from organic-rich black muds immediately underlying intertidal laminated microbial mats dominated by Microcoleus chthonoplastes yielded large, variable diameter spirochetes. These unusual spirochetes, previously reported only from the Alfacs Peninsula at the delta of the Ebro river in northeast Spain, contain striking arrays of cytoplasmic granules packed into their protoplasmic cylinders. On several occasions, both in summer and winter, the huge spirochetes were recognized in samples from mats growing in the Sippewissett salt marsh at Woods Hole Massachusetts. They were also seen in similar samples from microbial mats at North Pond, Laguna Figueroa, Baja California Norte, Mexico. The identity of these spirochetes was confirmed by electron microscopy: number and disposition of flagella, composite structure, measurements of their distinctive cytoplasmic granules. The granules, larger, more conspicuous and present in addition to ribosomes, are hypothesized to contain ATPases. As culture conditions worsen, these spirochetes retract into membrane-bounded round bodies in which they form refractile inclusions. From morphology and behavior we conclude the North American spirochetes from both Atlantic and Pacific intertidal microbial mats are indistinguishable from those at the delta of the Ebro river. We conclude a cosmopolitan distribution for Spirosymplokos deltaeiberi.

Histaminergic and non-histamine-immunoreactive mast cells within the cat lateral geniculate complex examined with light and electron microscopy

Mast cells and their location in the cat lateral geniculate complex of the thalamus were examined by means of histamine immunohistochemistry and the mast cell stain pinacyanol erythrosinate. Brain sections from seven normal adult pigmented cats were processed for light or electron microscopy. Histamine-containing and pinacyanol erythrosinate-stained mast cells were widespread throughout the dorsal and ventral lateral geniculate nuclei and the surrounding regions. Mast cells were especially numerous rostrally in the complex and in the geniculate C laminae. The cells were found consistently in association with blood vessels, ranging from capillary size to vessels c. 150 μm diameter, and twice as often with arterioles as with venules. Large clusters of many mast cells associated with single blood vessels were seen. Individual mast cells were typically 8 μm in diameter and somewhat oval, although multipolar and crescent-shaped cells were also seen, up to twice as long. The amount of histamine labeling varied across cells. When histamine-labeled material was secondarily stained with pinacyanol erythrosinate, many mast cells were double labeled. In addition, there was a small population of mast cells that stained only with pinacyanol erythrosinate, but was otherwise identical to the histamine-immunoreactive mast cells. Electron microscopic examination showed that the mast cells lie on the brain side of the blood-brain barrier. Mast cells were found in close proximity to the thalamic neuropil, primarily apposed to the processes of astrocytes, but also apposed to neural elements. The distinctive electron-dense cytoplasmic granules in the fully granulated, mature state were largely amorphous in appearance and as large as 700 nm in diameter. Histamine was dispersed throughout some granules and contained within restricted areas of other granules. In degranulated mast cells, large, irregularly shaped, electron-lucent granules were seen fused with the cell membrane on the neuropil side, as well as the lumen side of the mast cell. More mast cells were observed at the electron microscopic level than were expected from the light level observations, which suggests that, despite the numbers of mast cells labeled, these results may still underestimate the total mast cell population present in this region of the thalamus. Mast cells, by their numbers, their distribution and the potent chemical substances they contain, may significantly influence vascular and neural function, directly and indirectly, in the cat lateral geniculate complex.

Sequence divergence yet conserved physical characteristics among the E4 proteins of cutaneous human papillomaviruses

Human papillomavirus (HPV) types 1, 2, and 4 together comprise the major cause of cutaneous papillomas in the general population. We have aligned the genomes of these three viruses by partial sequence analysis, and have sequenced que E4 open reading frames (ORFs) of HPV 2 and HPV 4. After expression as ,β-gal fusion proteins in bacteria, antibodies raised to the putative E4 gene-products of both virus types were used to identify the native E4 proteins in naturally occurring tumors. At the primary amino acid sequence level, the E4 protein of HPV 2 was found to be most homologous with those of HPV 6 and 11 and was not closely related to those of HPV 1 or 4. Although the E4 ORF represents a region of weak homology amongst papillomaviruses, the E4 encoded proteins showed significant conservation in their physical characteristics. Like those of HPV 1, the E4 proteins of both HPV 2 and HPV 4 were found to be composed of a major low-molecular-weight doublet (16.5/18K for HPV 2, 20/21K for HPV 4, c.f. 16/17K for HPV 1) along with minor high-molecular-weight species, which probably represent dimers of the smaller proteins, (33K for HPV 2, 40K for HPV 4, c.f. 32/34K for HPV 1). The E4 products of all three virus types were multiply charged, and exhibited a characteristic migration pattern following alkaline urea gel electrophoresis. Although the levels of E4 expression in tumors induced by the different virus types was very different, this was found to correlate closely with the level of virus production characteristic of each virus type. In all three cases, E4 proteins were found to be primarily cytoplasmic, and to be associated with the distinctive cytoplasmic inclusion granules characteristic of each virus type. The poor sequence conservation between the E4 protein of HPVs 1, 2, and 4, taken alongside the ability of these viruses to infect similar histological sites, suggests that E4 may not be involved in determining tissue specificity. Our results suggest conserved physical characteristics (acidic, multiply charged, ability to form dimers) and similar site of expression may be the important factors for E4 function.