in vivo effects of iron-overload on asialoglycoprotein receptor (asgp-r) transcription

In vivoIn vivo effects of effects of iron-overload on iron-overload on

asialoglycoprotein asialoglycoprotein receptor (ASGP-R) receptor (ASGP-R)

transcriptiontranscriptionByBy

Giovanni CastorGiovanni Castor

5/05/2004 California State University Long Beach

Background information on Background information on ASGP-RASGP-R

Discovered in the 1970s by Ashwell and Discovered in the 1970s by Ashwell and MorellMorell




First reported mammalian lectinFirst reported mammalian lectin




First reported mammalian lectinFirst reported mammalian lectin

C-type lectin in mammalian liversC-type lectin in mammalian livers


Structure of ASGP-RStructure of ASGP-R Composed of major Composed of major

and minor subunitsand minor subunits



and minor subunitsand minor subunits Humans (2 subunits)Humans (2 subunits)



and minor subunitsand minor subunits Humans (2 subunits)Humans (2 subunits) Rats (3 subunits)Rats (3 subunits)



and minor subunitsand minor subunits Humans (2 subunits)Humans (2 subunits) Rats (3 subunits)Rats (3 subunits) 80% homology in 80% homology in

major subunitsmajor subunits



and minor subunitsand minor subunits Humans (2 subunits)Humans (2 subunits) Rats (3 subunits)Rats (3 subunits) 80% homology in 80% homology in

major subunitsmajor subunits 58% homology in 58% homology in

minor subunitsminor subunits


Known functions of Known functions of ASGP-RASGP-R

Recognizes glycoproteins Recognizes glycoproteins with a terminal galactose with a terminal galactose (Gal) or N-(Gal) or N-acetylgalactosamine (Gal-acetylgalactosamine (Gal-NAc) residues on the N-NAc) residues on the N-terminus of the terminus of the carbohydrate chaincarbohydrate chain


Known functions of Known functions of ASGP-RASGP-R

Recognizes glycoproteins Recognizes glycoproteins with a terminal galactose with a terminal galactose (Gal) or N-(Gal) or N-acetylgalactosamine (Gal-acetylgalactosamine (Gal-NAc) residues on the N-NAc) residues on the N-terminus of the terminus of the carbohydrate chaincarbohydrate chain

Investigators have found Investigators have found that ASGP-R can bind that ASGP-R can bind other ligandsother ligands


Significance & Significance & ImplicationsImplications

Over 35 years have passed since the Over 35 years have passed since the discovery of ASGP-R and the true discovery of ASGP-R and the true in in vivovivo functions have not been functions have not been discovereddiscovered




Selective targeting of Selective targeting of chemotherapeutic agents and chemotherapeutic agents and foreign genesforeign genes




Selective targeting of Selective targeting of chemotherapeutic agents and chemotherapeutic agents and foreign genesforeign genes

Implicated as a site of hepatitis B Implicated as a site of hepatitis B virus uptakevirus uptake


Preliminary StudyPreliminary Study

It was found that iron-overload of It was found that iron-overload of rats increased translation of ASGP-R rats increased translation of ASGP-R by 2-3 fold as compared to control by 2-3 fold as compared to control ratsrats


Question and HypothesisQuestion and Hypothesis

Does Does in vivoin vivo iron-overloading of rats iron-overloading of rats alter the transcription of ASGP-R?alter the transcription of ASGP-R?


Question and HypothesisQuestion and Hypothesis

Does Does in vivoin vivo iron-overloading of rats iron-overloading of rats alter the transcription of ASGP-R?alter the transcription of ASGP-R?

We hypothesize that the iron-We hypothesize that the iron-overload will cause an increase in overload will cause an increase in transcription of all subunits of ASGP-transcription of all subunits of ASGP-RR


Testing our hypothesisTesting our hypothesis Subjects: male Subjects: male

Sprague-Dawley rats Sprague-Dawley rats (AWB: 339001)(AWB: 339001)




Iron overload will be Iron overload will be induced by injection induced by injection of iron-dextran of iron-dextran




Iron overload will be Iron overload will be induced by injection induced by injection of iron-dextran of iron-dextran

2 Stages2 Stages


Stage 1: Creation of Stage 1: Creation of cDNA probescDNA probes

Isolation of total RNAIsolation of total RNA



Isolation of total RNAIsolation of total RNA RT-PCRRT-PCR



Isolation of total RNAIsolation of total RNA RT-PCRRT-PCR Cloning of PCR products via Cloning of PCR products via

plasmidsplasmids



Isolation of total RNAIsolation of total RNA RT-PCRRT-PCR Cloning of PCR products via Cloning of PCR products via

plasmidsplasmids PCR amplication of probe cDNA and PCR amplication of probe cDNA and

isotopic labeling of probeisotopic labeling of probe


Stage 2: Northern Blot Stage 2: Northern Blot analysisanalysis

Identification of increase or Identification of increase or decrease of ASGP-R mRNAdecrease of ASGP-R mRNA


Expected ResultsExpected Results

Anticipate data to support our Anticipate data to support our hypothesishypothesis



Anticipate data to support our Anticipate data to support our hypothesishypothesis

Distinct differences in our Northern Distinct differences in our Northern BlotsBlots


Concluding StatementConcluding Statement

The The in vivoin vivo function of ASGP-R has function of ASGP-R has yet to be determinedyet to be determined




Cell surface lectins have been Cell surface lectins have been implicated in bacterial and viral implicated in bacterial and viral infectioninfection




Cell surface lectins have been Cell surface lectins have been implicated in bacterial and viral implicated in bacterial and viral infectioninfection

Lead to treatments of diseasesLead to treatments of diseases


AcknowledgementsAcknowledgements

Howard Hughes Medical InstituteHoward Hughes Medical Institute California State University Long California State University Long

BeachBeach Dr. Douglas McAbeeDr. Douglas McAbee Dr. A.Z. MasonDr. A.Z. Mason

in vivo effects of iron-overload on asialoglycoprotein receptor (asgp-r) transcription

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