Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis.


BACKGROUND: Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts. RESULTS: B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion. CONCLUSIONS: These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.





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Kuan, Chien-Tsun, Jinli Chang, Jan-Eric Mansson, Jianjun Li, Charles Pegram, Pam Fredman, Roger E McLendon, Darell D Bigner, et al. (2010). Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis. BMC Dev Biol, 10. p. 114. 10.1186/1471-213X-10-114 Retrieved from

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Chien-Tsun Kuan

Adjunct Associate Professor in the Department of Pathology

Research Interests:
Conventional therapy for malignant brain tumors is ineffective. Targeted therapy using tumor-specific antibodies (MAb) alone or MAbs armed with radionuclides or toxins is a promising alternative approach for increasing therapeutic efficacy and decreasing toxicity to normal tissue. The major factors that influence antibody-targeted therapy for cancer treatment, including glioma therapy, are specificity, affinity, tumor penetration, toxicity and immunogenicity. The effective use of radioimmunotherapy (RAIT) for the treatment of solid malignancies has been limited by inadequate tumor penetration and non-targeted myelotoxicity resulting from the presence of radioimmunoconjugates in circulation. We believe that these limitations to direct RAIT can be overcome by using smaller engineered antibody-based molecules as vehicles and by selecting therapeutic radioisotopes with physical properties that complement the pharmacokinetics and pharmacodynamics of the antibody.

Our research is focused upon exploiting engineered antibody fragments to treat brain tumors by targeting to glioma-associated, oncofetal epitopes such as tenascin, glioma variant epidermal growth factor variant III (EGFRvIII), medulloblastoma-associated developmental markers, as well as the newly identified glioma-associated antigens, GPNMB and MRP3, by serial analysis of gene expression (SAGE). Projects performed in the current years have: 1) produced and evaluated the monovalent single-chain Fv (scFv) against EGRvIII in athymic mice bearing human glioma xenografts; 2) begun the development of divalent form of scFv, including diabody and minibody, to increase the efficacy of therapeutic agents in vivo; 3) generated CH2 domain-deleted Ch81C6 vs tenascin and evaluated the pharmacokinetics in mice and canines; 4) begun an extensive analysis of GPNMB and MRP3 protein expression correlated with measurement of RNA transcript levels and degree of DNA amplification.

Unarmed antibody can be effective against both subcutaneous and intracranial tumor models. The unarmed antibody approach with Mab Y10 vs EGFRvIII is very similar to the successful use of HerceptinTM. The mechanism is most likely both a direct antiproliferative effect with the induction of apoptosis and an indirect effect through the mobilization of antibody-mediated immune effector functions, such as complement and antibody-dependent cell-mediated cytotoxicity (ADCC). We also have begun to construct human/mouse chimeric Y10 to reduce immunogenicity of the Mab reagent and possibly enhance ADCC.

Our objectives for the coming years are to continue the optimization of engineered-antibody systems for in vivo application, namely; a) development of human/mouse chimeric anti-EGFRvIII murine Y10 with the same affinity and specificity but reduced immunogenicity and enhanced ADCC for in vivo application; b) to generate a totally human scFv specific to EGFRvIII but with anti-proliferative activity via screening from human phage libraries; c) generation of monomeric and dimeric anti-GPNMB/MRP3 scFvs and construction of immunoconjugate toxins or radiolabeled to determine the efficacy of therapeutic reagents in athymic rodent in athymic rodent in vivo models of intracranial glioma.


Roger Edwin McLendon

Professor of Pathology

Brain tumors are diagnosed in more than 20,000 Americans annually. The most malignant neoplasm, glioblastoma, is also the most common. Similarly, brain tumors constitute the most common solid neoplasm in children and include astrocytomas of the cerebellum, brain stem and cerebrum as well as medulloblastomas of the cerebellum.  My colleagues and I have endeavored to translate the bench discoveries of genetic mutations and aberrant protein expressions found in brain tumors to better understand the processes involved in the etiology, pathogenesis, and treatment of brain tumors.  Using the resources of the Preston Robert Brain Tumor Biorepository at Duke, our team, consisting of Henry Friedman, Allan Friedman, and Hai Yan and lead by Darell Bigner, have helped to identify mutations in Isocitrate Dehydrogenase (IDH1 and IDH2) as a marker of good prognosis in gliomas of adults.  This test is now offered at Duke as a clinical test.  Working with the Molecular Pathology Laboratory at Duke, we have also brought testing for TERT promoter region mutations as another major test for classifying gliomas in adults.  Our collaboration with the Toronto Sick Kids Hospital has resulted in prognostic testing for childhood medulloblastomas, primitive neuroectodermal tumors, and ependymomas at Duke.


Darell Doty Bigner

E. L. and Lucille F. Jones Cancer Distinguished Research Professor, in the School of Medicine

The Causes, Mechanisms of Transformation and Altered Growth Control and New Therapy for Primary and Metastatic Tumors of the Central Nervous System (CNS).

There are over 16,000 deaths in the United States each year from primary brain tumors such as malignant gliomas and medulloblastomas, and metastatic tumors to the CNS and its covering from systemic tumors such as carcinoma of the lung, breast, colon, and melanoma. An estimated 80,000 cases of primary brain tumors were expected to be diagnosed last year. Of that number, approximately 4,600 diagnosed will be children less than 19 years of age. During the last 20 years, however, there has been a significant increase in survival rates for those with primary malignant brain tumors.

For the last 44 years my research has involved the investigation of the causes, mechanism of transformation and altered growth control, and development of new methods of therapy for primary brain tumors and those metastasizing to the CNS and its coverings. In collaboration with my colleagues in the Preston Robert Tisch Brain Tumor Center, new drugs and those not previously thought to be active against CNS tumors have been identified. Overcoming mechanisms of drug resistance in primary brain tumors are also being pursued.

As the founding Director of the Preston Robert Tisch Brain Tumor Center, I help coordinate the research activities of all 37 faculty members in the Brain Tumor Center. These faculty members have projects ranging from very basic research into molecular etiology, molecular epidemiology, signal transduction; translational research performing pre-clinical evaluation of new therapies, and many clinical investigative efforts. I can describe any of the Brain Tumor Center faculty member’s research to third year students and then direct them to specific faculty members with whom the students would like a discussion.

We have identified through genome-wide screening methodology several new target molecules selectively expressed on malignant brain tumors, but not on normal brain. These include EGFRwt, EGFRvIII, and two lacto series gangliosides, 3'-isoLM1 and 3',6'-isoLD1 and chondroitin proteoglycan sulfate. We raised conventional and fully human monoclonal antibodies against most of these targets as well as having developed single fragment chain molecules from naïve human libraries.

My personal research focuses on molecularly targeted therapies of primary and metastatic CNS tumors with monoclonal antibodies and their fragments. Our study we conducted was with a molecule we discovered many years ago, the extracellular matrix molecule, Tenascin. We have treated over 150 malignant brain tumor patients with excellent results with a radiolabeled antibody we developed against Tenascin. We are collaborating with Dr. Ira Pastan at NIH to develop tumor-targeted therapies by fusing single fragment chain molecules from monoclonal antibodies or from naïve human libraries to the truncated fragment of pseudomonas exotoxin A. One example of this is the pseudomonas exotoxin conjugated to a single fragment chain antibody that reacts with wild type EGFR and EGFRvIII, two overexpressed proteins on glioblastoma. The immunotoxin, called D2C7-IT, is currently being investigated in an FDA dose-escalation study, in which patients undergoing treatment of this investigational new drug are showing positive responses. My laboratory is also working with Matthias Gromeier, creator of the oncolytic poliovirus - a re-engineered poliovirus that is lethal to cancer cells, but not lethal to normal cells. The oncolytic poliovirus therapeutic approach has shown such promising results in patients with glioblastoma, that it was recently featured on a on a special two-segment program of 60 Minutes. The next clinical step will be to combine both the virus and the immunotoxin with anti-PD1, an immune checkpoint blockade inhibitor and with anti-CD40, a fully human monoclonal antibody which converts tumor stimulant macrophages into tumor suppressive macrophages. We believe that regional tumor-targeted cytotoxic therapies, such as oncolytic poliovirus and the D2C7 immunotoxin, not only specifically target and destroy tumor cells, but in the process, initiate immune events that promote an in situ vaccine effect. That immune response can be amplified by human checkpoint blockade to engender a long-term systemic immune response that effectively eliminates recurrent and disseminated GBM cells. Ultimately, all three agents may be used together, providing different antigenic targets and cytotoxicity mechanisms.

We have identified through genome-wide screening methodology several new target molecules selectively expressed on malignant brain tumors, but not on normal brain. These include glycoprotein non-metastatic B (GPNMB), a molecule shared with malignant melanoma; MRP3, a member of the multidrug resistant family; and two lacto series gangliosides, 3'-isoLM1 and 3',6'-isoLD1 and chondroitin proteoglycan sulfate. We are raising conventional monoclonal antibodies against all of these targets as well as developing single fragment chain molecules from naïve human libraries. When necessary, affinity maturation in vitro is carried out and the antibodies and fragments are armed either with radioactive iodine, radioactive lutetium, or radioactive Astatine-211. Other constructs are evaluated for unarmed activity and some are armed with Pseudomonas exotoxin. After development of the constructs, they are evaluated in human malignant glioma xenograft systems and then all studies necessary for Investigational New Drug Permits from the Food and Drug Administration are carried out prior to actual clinical trial.

I was senior author on a New England Journal of Medicine paper that was the first to show markedly increased survival in low to intermediate grade gliomas with an isocitrate dehydrogenase mutation.

The first fully funded Specialized Research Center on Primary and Metastatic Tumors to the CNS funded by the National Institutes of Health, of which I was Principal Investigator, was funded for 30 years at which time the type of grant was discontinued. My NCI MERIT Award, which ranked in the upper 1.2 percentile of all NIH grants at the time of its last review, is currently in its 40th year of continuous funding. It is one of the few MERIT awards awarded three consecutive times, and it is the longest continually funded grant of the NCI Division of Cancer Diagnosis and Treatment. My last NCI Award was an Outstanding Investigator Award from 2014 to 2022.

In addition to the representative publications listed, I have made national presentations and international presentations during the past year.

My laboratory has trained over 50 third year medical students, residents, Ph.D. students, and postdoctoral fellows and I have a great deal of experience in career development with some students having advanced all the way from fellowship status to endowed professorships. A major goal with third year medical students is to perform work that can be presented in abstract form at national or international meetings and to obtain publication in major peer-reviewed journals.

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