Microtubule-associated protein, MAP1B, encodes functionally distinct polypeptides.

Microtubule-associated protein, MAP1B, is crucial for neuronal morphogenesis and disruptions in MAP1B function are correlated with neurodevelopmental disorders. MAP1B encodes a single polypeptide that is processed into discrete proteins, a heavy chain (HC) and a light chain (LC); however, it is unclear if these two chains operate individually or as a complex within the cell. In vivo studies have characterized the contribution of MAP1B HC and LC to microtubule and actin-based processes, but their molecular mechanisms of action are unknown. Using in vitro reconstitution with purified proteins, we dissect the biophysical properties of the HC and LC and uncover distinct binding behaviors and functional roles for these MAPs. Our biochemical assays indicate that MAP1B HC and LC do not form a constitutive complex, supporting the hypothesis that these proteins operate independently within cells. Both HC and LC inhibit the microtubule motors, kinesin-3, kinesin-4, and dynein, and differentially affect the severing activity of spastin. Notably, MAP1B LC binds to actin filaments in vitro and can simultaneously bind and crosslink actin filaments and microtubules, a function not observed for MAP1B HC. Phosphorylation of MAP1B HC by DYRK1a negatively regulates its actin-binding activity without significantly affecting its microtubule-binding capacity, suggesting a dynamic contribution of MAP1B HC in cytoskeletal organization. Overall, our study provides new insights into the distinct functional properties of MAP1B HC and LC, underscoring their roles in coordinating cytoskeletal networks during neuronal development.

Conserved roles for the dynein intermediate chain and Ndel1 in assembly and activation of dynein.

Processive transport by the microtubule motor cytoplasmic dynein requires the regulated assembly of a dynein-dynactin-adapter complex. Interactions between dynein and dynactin were initially ascribed to the dynein intermediate chain N-terminus and the dynactin subunit p150Glued. However, recent cryo-EM structures have not resolved this interaction, questioning its importance. The intermediate chain also interacts with Nde1/Ndel1, which compete with p150Glued for binding. We reveal that the intermediate chain N-terminus is a critical evolutionarily conserved hub that interacts with dynactin and Ndel1, the latter of which recruits LIS1 to drive complex assembly. In additon to revealing that the intermediate chain N-terminus is likely bound to p150Glued in active transport complexes, our data support a model whereby Ndel1-LIS1 must dissociate prior to LIS1 being handed off to dynein in temporally discrete steps. Our work reveals previously unknown steps in the dynein activation pathway, and provide insight into the integrated activities of LIS1/Ndel1 and dynactin/cargo-adapters.

Differential requirement for BRCA1-BARD1 E3 ubiquitin ligase activity in DNA damage repair and meiosis in the Caenorhabditis elegans germ line.

The tumor suppressor BRCA1-BARD1 complex regulates many cellular processes; of critical importance to its tumor suppressor function is its role in genome integrity. Although RING E3 ubiquitin ligase activity is the only known enzymatic activity of the complex, the in vivo requirement for BRCA1-BARD1 E3 ubiquitin ligase activity has been controversial. Here we probe the role of BRCA1-BARD1 E3 ubiquitin ligase activity in vivo using C. elegans. Genetic, cell biological, and biochemical analyses of mutants defective for E3 ligase activity suggest there is both E3 ligase-dependent and independent functions of the complex in the context of DNA damage repair and meiosis. We show that E3 ligase activity is important for nuclear accumulation of the complex and specifically to concentrate at meiotic recombination sites but not at DNA damage sites in proliferating germ cells. While BRCA1 alone is capable of monoubiquitylation, BARD1 is required with BRCA1 to promote polyubiquitylation. We find that the requirement for E3 ligase activity and BARD1 in DNA damage signaling and repair can be partially alleviated by driving the nuclear accumulation and self-association of BRCA1. Our data suggest that in addition to E3 ligase activity, BRCA1 may serve a structural role for DNA damage signaling and repair while BARD1 plays an accessory role to enhance BRCA1 function.

Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21.

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

Mucin 21 confers resistance to apoptosis in an O-glycosylation-dependent manner.

Highly glycosylated mucins protect epithelial surfaces from external insults and are related to malignant behaviors of carcinoma cells. However, the importance of carbohydrate chains on mucins in the process of cellular protection is not fully understood. Here, we investigated the effect of human mucin-21 (MUC21) expression on the susceptibility to apoptosis. MUC21 transfection into HEK293 cells decreased the number of apoptotic cells in culture media containing etoposide or after ultraviolet light irradiation. We used Chinese hamster ovary (CHO) cell variants to investigate the importance of MUC21 glycosylation in the resistance to apoptosis. When MUC21 was expressed in CHO-K1 cells, it was glycosylated with sialyl T-antigen and the cells showed resistance to etoposide-induced apoptosis. MUC21 transfection into Lec2 cells, a variant of CHO cells lacking sialylation of glycans, revealed that the presence of nonsialylated T-antigen also renders cells resistant to etoposide-induced apoptosis. MUC21 was transfected into ldlD cells and the glycosylation was manipulated by supplementation to the medium. Nonsupplemented cells and cells supplemented with N-acetylgalactosamine showed no resistance to etoposide-induced apoptosis. In contrast, these cells supplemented with N-acetylgalactosamine plus galactose expressed sialyl T-antigen and exhibited resistance to etoposide-induced apoptosis. Finally, galectin-3 knockdown in MUC21 transfectants of HEK293 cells did not significantly affect MUC21-dependent induction of apoptosis resistance. The results suggest that T-antigen with or without sialic acid is essential to the antiapoptotic effect of MUC21.

Association between Enterocyte Injury and Mortality in Patients on Hemodialysis Who Underwent Cardiac Surgery: An Exploratory Study.

BACKGROUND: Intestinal ischemia and enterocyte injury are significant causes of death after cardiac surgery. Hemodialysis is a well-known risk factor for intestinal ischemia. However, the relationship between enterocyte injury and mortality is unclear. This exploratory study assessed the association between intestinal fatty acid-binding protein (I-FABP), a specific marker of enterocyte injury, at intensive care unit (ICU) admission and in-hospital mortality in patients on hemodialysis who underwent cardiac surgery with cardiopulmonary bypass. MATERIALS AND METHODS: Forty-seven consecutive patients on long-term hemodialysis who underwent elective cardiac surgery (median age, 70 y; men, 27 [57%]) were prospectively enrolled. The association between serum I-FABP levels at ICU admission and in-hospital mortality was compared with the associations between serum I-FABP levels and prognostic severity scores, vasoactive-inotropic scores, and lactate levels. RESULTS: Only I-FABP levels at ICU admission were significantly related to in-hospital mortality (odds ratio, 5.54; 95% confidence interval [CI], 1.08-28.43) in the simple logistic regression analysis. Univariate and multiple linear regression analyses indicated prolonged cardiopulmonary bypass (ρ, 0.49; 95% CI, 0.15-0.83), higher mean norepinephrine dose (ρ, 0.07; 95% CI, 0.02-0.12), lower mean dopamine dose (ρ, -0.51; 95% CI, -0.94 to -0.08), and intra-aortic balloon pump use (ρ, 3.63; 95% CI, 1.68-5.59) were significant risk factors for high I-FABP levels. CONCLUSIONS: Enterocyte injury at ICU admission was associated with in-hospital mortality after cardiac surgery for patients on hemodialysis. Intraoperative hidden hypoperfusion of the intestine may impact prognoses. Enterocyte injury prevention, early diagnosis, and intervention for intestinal ischemia might be required to improve outcomes.

Intestinal fatty acid-binding protein levels in patients with chronic renal failure.

BACKGROUND: Intestinal fatty acid-binding protein (I-FABP), a biomarker of enterocyte injury, has been reported to be a diagnostic marker of intestinal ischemia and a prognostic marker in critically ill patients. However, the kinetics of I-FABP in renal failure patients is unknown. We sought to identify I-FABP levels in patients with chronic kidney disease (CKD) and end-stage kidney disease (ESKD) on hemodialysis (HD) and to identify the manner in which the I-FABP levels change. MATERIALS AND METHODS: Adult patients who were admitted for elective cardiac surgery with either normal renal function (NRF), CKD, or ESKD on HD were enrolled. Serum I-FABP levels in NRF and CKD patients and in ESKD patients before and after HD were determined. RESULTS: A total of 124 patients were evaluated: 47 NRF, 53 CKD, and 24 ESKD. The I-FABP levels of the CKD patients and pre-HD ESKD patients were significantly higher than those of the NRF patients (P = 0.018 and P <0.001, respectively). I-FABP levels were significantly negatively correlated with the estimated glomerular filtration rate in NRF and CKD patients (Spearman's ρ = -0.313, P = 0.002). In addition, I-FABP levels in ESKD patients were significantly lower after HD than those before HD (P <0.001). CONCLUSIONS: I-FABP levels in CKD and pre-HD ESKD patients were significantly higher than those in NRF patients. In addition, I-FABP was significantly eliminated by HD in patients with ESKD. Clinicians and researchers should consider this aspect of I-FABP when using it as a diagnostic and prognostic marker in patients with renal insufficiency.

High-resolution monochromator for iron nuclear resonance vibrational spectroscopy of biological samples.

A new high-resolution monochromator for 14.4-keV X-rays has been designed and developed for the Fe nuclear resonance vibrational spectroscopy of biological samples. Higher flux and stability are especially important for measuring biological samples, because of the very weak signals produced due to the low concentrations of Fe-57. A 24% increase in flux while maintaining a high resolution below 0.9 meV is achieved in the calculation by adopting an asymmetric reflection of Ge, which is used as the first crystal of the three-bounce high-resolution monochromator. The small cost resulting from a 20% increase of the exit beam size is acceptable to our biological applications. The higher throughput of the new design has been experimentally verified. A fine rotation mechanics that that combines a weak-link hinge with a piezoelectric actuator was used for controlling the photon energy of the monochromatic beam. The resulting stability is sufficient to preserve the intrinsic resolution.

Pineal cyst resection in the absence of ventriculomegaly or Parinaud's syndrome: clinical outcomes and implications for patient selection.

OBJECT: Surgical indications for patients with pineal cysts are controversial. While the majority of patients harboring a pineal cyst require no treatment, surgery is a well-accepted option for a subset of those patients with secondary hydrocephalus or Parinaud's syndrome. The majority of pineal cysts are identified incidentally during workup for other potential conditions, which may or may not be related to the presence of the cyst. In the absence of clear obstruction of CSF pathways, the treatment of presumed symptomatic pineal cysts is debatable. To clarify the role of surgery in these borderline cases, the authors reviewed their experience with resection of pineal cysts in the absence of ventriculomegaly or Parinaud's syndrome. METHODS: The authors retrospectively reviewed medical records and imaging of all patients surgically treated between 2001 and 2014 with a pineal cyst in the absence of ventriculomegaly and Parinaud's syndrome. The presenting symptoms, preoperative cyst size, preoperative radiographic aqueductal compression, extent of resection, and radiographic and clinical follow-up were documented. RESULTS: Eighteen patients (14 female and 4 male; mean age 24 years, range 4-47 years) underwent cyst resection in the absence of ventriculomegaly or Parinaud's syndrome. Presenting symptoms included headache (17 patients), visual disturbances (10 patients), gait instability (5 patients), dizziness (5 patients), episodic loss of consciousness (2 patients), and hypersomnolence (1 patient). The mean preoperative cyst diameter was 1.5 cm (range 0.9-2.2 cm). All patients had a complete resection. At a mean clinical follow-up of 19.1 months (range postoperative to 71 months), 17 (94%) patients had resolution or improvement of their presenting symptoms. CONCLUSIONS: The authors' results suggest that ventriculomegaly and Parinaud's syndrome are not absolute requisites for a pineal cyst to be symptomatic. Analogous to colloid cysts of the third ventricle, intermittent occlusion of cerebrospinal fluid pathways may cause small pineal cysts to become intermittently symptomatic. A select cohort of patients with pineal cysts may benefit from surgery despite a lack of hydrocephalus or other obvious compressive pathology.

Structural mechanism of the dynein power stroke.

Dyneins are large microtubule motor proteins required for mitosis, intracellular transport and ciliary and flagellar motility. They generate force through a power-stroke mechanism, which is an ATP-consuming cycle of pre- and post-power-stroke conformational changes that cause relative motion between different dynein domains. However, key structural details of dynein's force generation remain elusive. Here, using cryo-electron tomography of intact, active (that is, beating), rapidly frozen sea urchin sperm flagella, we determined the in situ three-dimensional structures of all domains of both pre- and post-power-stroke dynein, including the previously unresolved linker and stalk of pre-power-stroke dynein. Our results reveal that the rotation of the head relative to the linker is the key action in dynein movement, and that there are at least two distinct pre-power-stroke conformations: pre-I (microtubule-detached) and pre-II (microtubule-bound). We provide three-dimensional reconstructions of native dyneins in three conformational states, in situ, allowing us to propose a molecular model of the structural cycle underlying dynein movement.

A case report of human immunodeficiency virus-associated anaplastic lymphoma kinase protein-negative anaplastic large cell lymphoma.

Human immunodeficiency virus (HIV)-associated anaplastic large cell lymphoma (ALCL) is not so common, and anaplastic lymphoma kinase protein (ALK)-negative ALCL is rare and has a low survival rate. We report a case of a 31-year-old Japanese man diagnosed with HIV-associated ALK-negative ALCL who presented with long-lasting fever of unknown origin. The diagnosis was based on a full work-up that included inguinal lymph-node biopsy. Eight-cycle chemotherapy that included cyclophosphamide, doxorubicin, vincristine, and prednisone in addition to antiretroviral therapy for HIV infection provided a complete remission of his ALCL and over 5-year survival for him.

Association between chronic hepatitis C virus infection and high levels of circulating N-terminal pro-brain natriuretic peptide.

The association between HCV infection and myocardial disorders remains unclear. This study aimed to assess whether or not HCV infection influences myocardial dysfunction by the use of NT-proBNP, a sensitive marker of myocardial dysfunction. A total of 198 participants [99 patients with chronic HCV infection (aged 46-68 years) and 99 anti-HCV-negative sex and age matched controls] were examined. Serum HCV-RNA level and HCV genotype were tested and liver biopsy was done only for the patient group. The NT-proBNP concentration of the HCV patients (mean 71.6 ± 79.1 pg/ml; median 46.0 pg/ml, range 5.0-400.0) was significantly higher than that of the controls (mean 39.8 ± 24.4 pg/ml; median 35.8 pg/ml, range 7.0-108.0) (P < 0.05). 20.0 % of the HCV patients and 0.6 % of the controls had high NT-proBNP (higher than 125 pg/ml; the single cut off point for patients under 75 years of age) (P < 0.05). Stepwise multiple regression analysis revealed that chronic HCV infection was independently correlated with NT-proBNP level after adjustment for parameters that might influence NT-proBNP (P = 0.005). Our data suggest that chronic HCV infection is associated with increased NT-proBNP, indicating that chronic HCV infection might induce myocardial dysfunction.

The serum undercarboxylated osteocalcin level and the diet of a Japanese population: results from the Kyushu and Okinawa Population Study (KOPS).

The elevated serum undercarboxylated osteocalcin (ucOC) level is related to osteoporosis. In Japan, vitamin K intake is lower, and the incidence of hip fractures noticeably higher in northern Kyushu than in other areas. The study was done to determine the serum ucOC levels in a Japanese population and its association with diet and glucose metabolism. The data of 3,658 healthy adults aged 40-69 (1,373 men and 2,285 women) who lived in northern Kyushu area were analyzed. The data included anthropometric measurements and a self-reported personal interview on daily intake of foods. The serum ucOC level of each participant was measured by electrochemiluminescence immunoassay. Glycohemoglobin A1c (HbA1c), fasting plasma glucose, and serum insulin concentrations were measured. The median serum ucOC level of the women (4.65 ng/mL) was significantly higher than that of the men (3.04 ng/mL) (P = 0.0021). The age-specific ucOC levels of the men decreased significantly with age. In contrast, the ucOC levels of the women aged ≥50 were elevated, but the levels varied markedly within the other age groups. For both men and women, multivariate analysis identified a daily diet rich in vitamin K and HbA1c level as independently having a significant, negative relationship to serum ucOC level. Our study indicates that the serum ucOC decreases with age in men, increases postmenopausally in women, and correlates inversely with dietary consumption of certain foods and with fasting glucose and HbA1c level.

Raloxifene hydrochloride is an adjuvant antiviral treatment of postmenopausal women with chronic hepatitis C: a randomized trial.

BACKGROUND & AIMS: Early menopause in women with chronic hepatitis C virus (HCV) infection is associated with a low likelihood of a sustained virological response (SVR) in conjunction with their antiviral treatment. This is potentially related to their reduced estrogen secretion. The study was done to determine whether selective estrogen receptor modulator administration might improve the efficacy of the current standard of care (SOC) treatment, pegylated interferon (PegIFN) α2a plus ribavirin (RBV), for postmenopausal women. METHODS: One hundred and twenty-three postmenopausal women with genotype 1b chronic hepatitis C were randomly assigned to one of two treatment groups: raloxifene hydrochloride (RLX) (60 mg/day) plus SOC (PegIFNα2a 180 µg/week and RBV 600-1,000 mg/day) (n=62) or SOC only (n=61). Genotyping was performed of the polymorphism in the interleukin-28B (IL28B) gene region (rs8099917) of DNA collected from each patient. RESULTS: One RLX-treated patient discontinued RLX because of a systemic rash following 2 weeks of treatment. Twenty-four weeks after treatment, the SVR rate was significantly higher for RLX plus SOC patients (61.3%) than for SOC only patients (34.4%) (p=0.0051). Further, the SVR rate was significantly higher for RLX plus SOC patients with IL28B TT (72.5%) than for SOC only patients with IL28B TT (39.2%) (p=0.0014), but no such relationship was observed in patients carrying the minor IL28B allele. CONCLUSIONS: RLX improved the efficacy of SOC in the treatment of postmenopausal women with chronic hepatitis C. RLX shows promise as an adjuvant to the standard antiviral treatment of such patients.

Mucin 21 in esophageal squamous epithelia and carcinomas: analysis with glycoform-specific monoclonal antibodies.

Monoclonal antibodies (mAbs) against mucin 21 (MUC21), a human counterpart of mouse epiglycanin/Muc21, were prepared using human embryonic kidney 293 cells transfected with MUC21 as the immunogen. The specificity of these mAbs was examined by flow cytometry, immunoprecipitation and western blotting focusing on the differential glycosylation of MUC21 expressed in variant Chinese hamster ovary (CHO) cells (ldlD cells and Lec2 cells) and CHO-K1 cells. One of these mAbs, heM21D, bound to both the unmodified core polypeptide of MUC21 and MUC21 attached with N-acetylgalactosamine (Tn-MUC21). Six antibodies, including mAb heM21C, bound to MUC21 with Tn, T or sialyl-T epitopes but not the unmodified core polypeptide of MUC21. Esophageal squamous carcinomas and adjacent squamous epithelia were immunohistochemically examined for the binding of these mAbs. MUC21 was expressed in esophageal squamous epithelial cells, and its O-glycan extended forms were observed in the luminal portions of squamous epithelia. As revealed by the binding of mAb heM21D and the absence of reactivity with mAb heM21C, esophageal squamous carcinoma cells produce MUC21 without the attachment of O-glycans. This is the first report to show that there is a change in the glycoform of MUC21 that can be used to differentiate between squamous epithelia and squamous carcinoma of the esophagus. Thus, these antibodies represent a useful tool to characterize squamous epithelial differentiation and carcinogenesis.

Quinolone derivatives containing strained spirocycle as orally active glycogen synthase kinase 3ß (GSK-3ß) inhibitors for type 2 diabetics.

The design, synthesis, and evaluation of 6-6-7 tricyclic quinolones containing the strained spirocycle moiety aiming at the GSK-3ß inhibitor were described. Among the synthesized compounds, 44, having a cyclobutane ring on a spirocycle, showed excellent GSK-3ß inhibitory activity in both cell-free and cell-based assays (IC(50) = 36nM, EC(50) = 3.2µM, respectively). Additionally, 44 decreased the plasma glucose concentration dose-dependently after an oral glucose tolerance test in mice.

Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1.

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal "basic" domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly-promoting factors with complementary activities.

Mucin 21/epiglycanin modulates cell adhesion.

The molecular structure of mouse Mucin 21 (Muc21)/epiglycanin is proposed to have 98 tandem repeats of 15 amino acids and three exceptional repeats with 12 or 13 amino acids each, followed by a stem domain, a transmembrane domain, and a cytoplasmic tail. A cDNA of Muc21 having 84 tandem repeats of 15 amino acids was constructed and transfected using a Venus vector into HEK 293T cells. The fluorescent cells, which were considered to express Muc21, were nonadherent. This antiadhesion effect was lessened when constructs with smaller numbers of tandem repeats were used, suggesting that the tandem repeat domain plays a crucial role. Cells expressing Muc21 were significantly less adherent to each other and to extracellular matrix components than control cells. Antibody binding to the cell surface integrin subunits alpha5, alpha6, and beta1 was reduced in Muc21 transfectants in a tandem repeat-dependent manner, whereas equal amounts of proteins were detected by Western blot analysis. Muc21 was expressed as a large glycoprotein that was highly glycosylated with O-glycans at the cell surface, as detected by flow cytometry, Western blotting, and lectin blotting. Although at least a portion of Muc21 was glycosylated with sialylated glycans, removal of sialic acid did not influence the prevention of adhesion.

Two-year comparison of photodynamic therapy and intravitreal bevacizumab for treatment of myopic choroidal neovascularisation.

AIM: To compare the long-term outcome of photodynamic therapy (PDT) with that of intravitreal bevacizumab (IVB) for myopic choroidal neovascularizations (mCNVs). METHODS: 24 eyes were selected from 40 consecutive patients with mCNV, and the patients were divided into Group A, consisting of 12 eyes treated by PDT, and Group B, consisting of 12 eyes treated by 1.25 mg IVB. The age and best-corrected visual acuity (BCVA) were matched between the two groups. The BCVA, size of the chorioretinal atrophy surrounding the CNV (CRA), central foveal thickness (CFT) and CNV thickness were determined before and at 12 and 24 months after the treatment. RESULTS: The BCVA did not change after PDT but was significantly improved from 0.75+/-0.25 to 0.49+/-0.42 logMAR units at 12 months and to 0.50+/-0.38 logMAR units at 24 months after IVB. The CFT were significantly reduced in both groups at 12 and 24 months. The CRAs were larger in group A than in group B at 12 and 24 months, and their sizes were correlated with the BCVA. CONCLUSION: At 24 months, IVB is more effective than PDT in treating mCNV. The enlargement of the CRA might be related to the incomplete visual recovery after PDT.

Identification and expression of human epiglycanin/MUC21: a novel transmembrane mucin.

The gene for the human orthologue of mouse epiglycanin, a mucin expressed on mammary carcinoma TA3-Ha cells but not TA3-St cells, was identified by homology search to a mouse epiglycanin cDNA fragment identified by representational difference analysis between TA3-Ha and TA3-St cells. The open reading frame of this gene was cloned from human cervical carcinoma ME-180 cells. It consists of a mucin domain with 28 nonidentical tandem repeats of 45 nucleotides each corresponding to a threonine/serine-rich peptide, a stem domain, a transmembrane domain, and a cytoplasmic tail. The cloned cDNA with a FLAG sequence was expressed in K562 cells. A combination of immunoprecipitation with a polyclonal antibody specific for the cytoplasmic tail and Western blotting analysis with an anti-FLAG antibody and lectins revealed a mucin-like component as the gene product. Analysis by the use of tissue cDNA libraries indicated that the gene is expressed in lung, large intestine, thymus, and testis among 16 normal tissues tested. The polyclonal antibody specific for a synthetic peptide from the cytoplasmic tail, when tested for its reactivity with normal lung tissues, reacted with epithelia of bronchi and bronchioli but not with alveoli. All of 24 lung adenocarcinomas specimens tested were reactive with the antibody, whereas reactivity was observed with only 2 out of 24 squamous and none out of 24 small cell lung carcinomas. This is a novel transmembrane mucin and designated as MUC21.