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

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

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

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

5/05/2004 California State University Long Beach

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

and minor subunitsand minor subunits

5/05/2004 California State University Long Beach

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

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

5/05/2004 California State University Long Beach

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

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

5/05/2004 California State University Long Beach

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

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

5/05/2004 California State University Long Beach

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

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

5/05/2004 California State University Long Beach

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

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

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

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

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

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

5/05/2004 California State University Long Beach

5/05/2004 California State University Long Beach

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?

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

Testing our hypothesisTesting our hypothesis Subjects: male Subjects: male

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

5/05/2004 California State University Long Beach

Testing our hypothesisTesting our hypothesis Subjects: male Subjects: male

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

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

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

2 Stages2 Stages

5/05/2004 California State University Long Beach

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

Isolation of total RNAIsolation of total RNA

5/05/2004 California State University Long Beach

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

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

5/05/2004 California State University Long Beach

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

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

plasmidsplasmids

5/05/2004 California State University Long Beach

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

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

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

Expected ResultsExpected Results

Anticipate data to support our Anticipate data to support our hypothesishypothesis

5/05/2004 California State University Long Beach

Expected ResultsExpected Results

Anticipate data to support our Anticipate data to support our hypothesishypothesis

Distinct differences in our Northern Distinct differences in our Northern BlotsBlots

5/05/2004 California State University Long Beach

Expected ResultsExpected Results

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

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

5/05/2004 California State University Long Beach

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

Lead to treatments of diseasesLead to treatments of diseases

5/05/2004 California State University Long Beach

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