Hepatocyte ASGR1/ASGR2 GalNAc-conjugate uptake and endosomal routing — clathrin/AP-2/DNM2 internalization, V-ATPase acidification, EHD1 receptor recycling vs. ESCRT/RAB7A late-endosomal maturation, CTSD lysosomal degradation, and cytosolic oligonucleotide escape

Hepatocyte-targeted N-acetylgalactosamine (GalNAc)-conjugated siRNA and antisense oligonucleotide therapeutics, as well as desialylated plasma glycoproteins, are selectively captured at the sinusoidal hepatocyte surface by the hetero-oligomeric asialoglycoprotein receptor complex (ASGR1/HL-1 and ASGR2/HL-2). Receptor-ligand complexes are concentrated into clathrin-coated pits by the AP-2 adaptor complex (AP2M1) and clathrin heavy chain (CLTC) and severed from the plasma membrane by the mechanochemical GTPase dynamin-2 (DNM2). Following vesicle uncoating, V-ATPase (ATP6V1A) acidifies the early sorting endosome lumen (pH ~6.0-5.5), triggering Ca2+ loss from the C-type lectin carbohydrate-recognition domains of ASGR1/ASGR2 and releasing the GalNAc-conjugate payload. From the acidified sorting endosome, flux partitions across three competing branches: (1) rapid tubular endocytic recycling of uncoupled ASGR1/ASGR2 back to the sinusoidal plasma membrane mediated by EHD1; (2) ESCRT-I (TSG101) multivesicular body sorting and RAB7A-driven early-to-late endosomal maturation, which delivers the vast majority of internalized GalNAc oligonucleotide and glycoprotein cargo to late endosomes and lysosomes for sequestration or hydrolytic/proteolytic turnover (represented by CTSD; PMID:12171918, PMID:29069408); and (3) a slow, rate-limiting endosomal membrane translocation step that releases a small productive fraction of oligonucleotide payload into the hepatocyte cytosol to engage RISC or RNase H1 (a minuscule fraction in vivo for GalNAc conjugates [PMID:29069408], modeled qualitatively as ~10% at baseline under uniform uncalibrated ABM transition rates with RAB7A-dependent late-endosomal competition extrapolated from hepatic LNP delivery [PMID:41814093]).

MODULE:asgr_galnac_hepatocyte_deliveryDRAFTTransport Stepmodules/asgr_galnac_hepatocyte_delivery.yaml

carbohydrate bindingGO:0030246 receptor-mediated endocytosisGO:0006898 endosomal lumen acidificationGO:0048388 endocytic recyclingGO:0032456 endosome to lysosome transportGO:0008333

GO:0030246

carbohydrate binding

Ca2+-dependent C-type lectin recognition of terminal galactose and GalNAc residues by ASGR1 and ASGR2.

GO:0006898

receptor-mediated endocytosis

Clathrin-, AP-2-, and dynamin-2-mediated internalization of ASGR-GalNAc cargo complexes at the hepatocyte surface.

GO:0048388

endosomal lumen acidification

V-ATPase-driven proton translocation lowers early endosomal pH to uncouple GalNAc ligands from ASGR1/ASGR2.

GO:0032456

endocytic recycling

EHD1-dependent tubular recycling returns ligand-free ASGR1/ASGR2 receptors to the hepatocyte plasma membrane.

GO:0008333

endosome to lysosome transport

ESCRT-I (TSG101) and RAB7A route retained endosomal cargo into late endosomes and lysosomes, competing with cytosolic escape.

PMID:29069408

Asialoglycoprotein receptor 1 mediates productive uptake of N-acetylgalactosamine-conjugated and unconjugated phosphorothioate antisense oligonucleotides into liver hepatocytes.

ASGR1 mediates high-affinity hepatocyte uptake of GalNAc-conjugated oligonucleotides via the clathrin-dependent endocytic pathway, while only a minuscule fraction of internalized oligonucleotide escapes endocytic compartments into the cytoplasm.

It is clear, however, that only a minuscule portion of endocytosed ASO escapes into the cytoplasmic/nuclear space to mediate target cleavage, while the vast majority remains trapped in endocytic compartments and is inactive (4,5).

PMID:41814093

In vivo endosomal escape assay identifies mechanisms for efficient hepatic LNP delivery.

In vivo hepatic endosomal escape is restricted by Rab7-mediated late endosomal maturation, and loss of Rab7 diverts endosomal cargo away from lysosomal sequestration to increase cytosolic delivery.

Loss of Rab7, a mediator of late endosomal maturation, increased LNP escape.

PMID:12171918

Nonpalmitoylated human asialoglycoprotein receptors recycle constitutively but are defective in coated pit-mediated endocytosis, dissociation, and delivery of ligand to lysosomes.

The human hepatic asialoglycoprotein receptor heterocomplex (ASGR1/H1 and ASGR2/H2) internalizes desialylated glycoproteins via the clathrin-coated pit pathway and delivers ligand to lysosomes.

The hepatic asialoglycoprotein receptor (ASGP-R) internalizes desialylated glycoproteins via the clathrin-coated pit pathway and mediates their delivery to lysosomes for degradation.

file:human/ASGR1/ASGR1-ai-review.yaml

ASGR1 gene review (human)

Major subunit of the hepatic asialoglycoprotein receptor mediating galactose/GalNAc binding and clathrin-dependent receptor-mediated endocytosis (UniProtKB:P07306, GO:0030246, GO:0006898).

file:human/ASGR2/ASGR2-ai-review.yaml

ASGR2 gene review (human)

Minor subunit of the hepatic asialoglycoprotein receptor complex (UniProtKB:P07307, GO:0030246, GO:0006898).

file:human/AP2M1/AP2M1-ai-review.yaml

AP2M1 gene review (human)

Tyrosine-motif cargo adaptor coupling plasma-membrane receptors to clathrin-coated pits (UniProtKB:Q96CW1, GO:0005048, GO:0072583).

file:worm/dpy-23/dpy-23-ai-review.yaml

dpy-23 gene review (C. elegans)

Orthologous metazoan AP-2 mu2 subunit recognizing YxxPhi motifs for clathrin-dependent receptor-mediated endocytosis (UniProtKB:P35603, GO:0035615, GO:0072583).

file:human/CLTC/CLTC-ai-review.yaml

CLTC gene review (human)

Clathrin heavy chain triskelion lattice assembly for coated-vesicle budding (UniProtKB:Q00610, GO:0005198, GO:0006898).

file:human/DNM2/DNM2-ai-review.yaml

DNM2 gene review (human)

Mechanochemical GTPase motor driving clathrin-coated vesicle neck scission (UniProtKB:P50570, GO:0061791, GO:0099050).

file:human/ATP6V1A/ATP6V1A-ai-review.yaml

ATP6V1A gene review (human)

V-ATPase catalytic subunit A driving endosomal lumen acidification (UniProtKB:P38606, GO:0046961, GO:0048388).

file:human/EHD1/EHD1-ai-review.yaml

EHD1 gene review (human)

Recycling endosome tubulation and fission ATPase returning receptors to the cell surface (UniProtKB:Q9H4M9, GO:0016887, GO:0032456).

file:human/TSG101/TSG101-ai-review.yaml

TSG101 gene review (human)

ESCRT-I subunit mediating MVB sorting (UniProtKB:Q99816, GO:0043130, GO:0036258).

file:human/RAB7A/RAB7A-ai-review.yaml

RAB7A gene review (human)

Small GTPase governing early-to-late endosome maturation and lysosome fusion (UniProtKB:P51149, GO:0003924, GO:0045022, GO:0008333).

file:mouse/Rab7/Rab7-ai-review.yaml

Rab7 gene review (mouse)

Mouse Rab7 mediates hepatic late endosomal maturation and restricts cytosolic payload escape (UniProtKB:P51150, PMID:41814093).

file:human/CTSD/CTSD-ai-review.yaml

CTSD gene review (human)

Lysosomal aspartic endopeptidase executing terminal degradative proteolysis (UniProtKB:P07339, GO:0004190, GO:0006508).

8Nodes

7Parts

0Variant Sets

0Variants

10Annotons

7Connections

Executable models all models

Agent-based derived from this module

Agent-based hepatocyte ASGR1/ASGR2 GalNAc-conjugate uptake, receptor recycling, lysosomal routing, and cytosolic escape model

✓ Wild-type hepatocyte GalNAc uptake (rapid receptor recycling + lysosomal sequestration dominate; ~10% cytosolic escape)
✓ RAB7A / late-endosomal maturation loss (blocks lysosomal delivery and increases cytosolic GalNAc payload escape)
✓ Clathrin / AP-2 / DNM2 internalization block (GalNAc conjugates trapped on sinusoidal ASGR1/ASGR2)
✓ V-ATPase acidification block (ATP6V1A loss / bafilomycin A1; arrest in neutral nascent vesicles)
✓ EHD1 recycling block (diverts sorting-endosome flux into lysosomal degradation and cytosolic escape)
✓ CTSD lysosomal proteolysis block (undegraded cargo accumulates in late endosomes/lysosomes)

Run in browser →

Derived QC

Recommended-field compliance

100.0% recommended fields populated

All recommended fields populated.

Module deep research

✗ none found

No MODULE:asgr_galnac_hepatocyte_delivery deep-research report alongside the module YAML.

Leaf nodes lacking representative members

1 leaf node(s) with no concrete protein grounding:

Productive cytosolic GalNAc-siRNA/ASO escape (RISC / RNase H1 engagement)

Template conformance

✓ every declared conforms_to bundle matches its template motif.

Function consistency and core coverage

11 gene-function check(s) reflected in core functions · 0 conflict(s) · 8 evidence or coverage gap(s) (23 comparison(s))

Retained noncore functions are reported separately; they do not require promotion to core functions. Support from representative genes does not establish conservation across the whole family. Missing reviews or identifiers leave a claim unverified.

Annoton	Participant	Function	Assessment
asgr1_lectin_capture	ASGR1 (human) UniProtKB:P07306 Family Representative	asialoglycoprotein receptor activity GO:0004873	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
asgr2_lectin_capture	ASGR2 (human) UniProtKB:P07307 Family Representative	asialoglycoprotein receptor activity GO:0004873	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ap2m1_cargo_selection	AP2M1 (human) UniProtKB:Q96CW1 Family Representative	signal sequence receptor activity GO:0005048	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ap2m1_cargo_selection	dpy-23 (C. elegans) UniProtKB:P35603 Family Representative	signal sequence receptor activity GO:0005048	Function Unreviewed No retained annotation or core molecular function supports this GO assertion. Missing or broader evidence is a coverage gap, not proof the activity is absent.
cltc_coat_lattice	CLTC (human) UniProtKB:Q00610 Family Representative	structural molecule activity GO:0005198	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
dnm2_vesicle_scission	DNM2 (human) UniProtKB:P50570 Family Representative	GTPase motor activity GO:0061791	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
atp6v1a_proton_pump	ATP6V1A (human) UniProtKB:P38606 Family Representative	proton-transporting ATPase activity, rotational mechanism GO:0046961	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ehd1_membrane_fission	EHD1 (human) UniProtKB:Q9H4M9 Family Representative	ATP hydrolysis activity GO:0016887	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
tsg101_escrt_sorting	TSG101 (human) UniProtKB:Q99816 Family Representative	ubiquitin binding GO:0043130	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
rab7a_late_maturation	RAB7A (human) UniProtKB:P51149 Family Representative	GTPase activity GO:0003924	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
rab7a_late_maturation	Rab7 (mouse) UniProtKB:P51150 Family Representative	GTPase activity GO:0003924	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ctsd_proteolysis	CTSD (human) UniProtKB:P07339 Family Representative	aspartic-type endopeptidase activity GO:0004190	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
asgr1_lectin_capture	Asialoglycoprotein receptor 1 PANTHER:PTHR22802:SF470 Family	asialoglycoprotein receptor activity GO:0004873	Family Supported Family review supports GO:0004873 for PANTHER:PTHR22802:SF470. Asialoglycoprotein receptor activity (GO:0004873) — Ca2+-dependent recognition and clathrin-mediated endocytic clearance of desialylated Gal/GalNAc-terminated plasma glycoproteins by hepatocytes — is specific to the hepatic asialoglycoprotein receptor subfamilies (SF470 ASGR1 and SF433 ASGR2, plus avian/non-mammalian hepatic lectin orthologs SF461 and SF454) and does not apply to myeloid mannose/PRR subfamilies (CD209, CLEC4E, CLEC6A) or IgE receptor FCER2.
asgr1_lectin_capture	Asialoglycoprotein receptor 1 PANTHER:PTHR22803:SF159 Family	asialoglycoprotein receptor activity GO:0004873	Family Supported Family review supports GO:0004873 for PANTHER:PTHR22803:SF159. Within PTHR22803, asialoglycoprotein receptor activity (Gal/GalNAc-specific clearance of desialylated serum glycoproteins) is restricted to SF159 (ASIALOGLYCOPROTEIN RECEPTOR 1; together with ASGR2, which carries a family-level UniProt DR PANTHER; PTHR22803 cross-reference while its PANTHER subfamily is PTHR22802:SF433) and is not shared by mannose receptors, phospholipase A2 receptors, eosinophil granule cytotoxins, or snaclecs.
asgr2_lectin_capture	Asialoglycoprotein receptor 2 PANTHER:PTHR22802:SF433 Family	asialoglycoprotein receptor activity GO:0004873	Family Supported Family review supports GO:0004873 for PANTHER:PTHR22802:SF433. Asialoglycoprotein receptor activity (GO:0004873) — Ca2+-dependent recognition and clathrin-mediated endocytic clearance of desialylated Gal/GalNAc-terminated plasma glycoproteins by hepatocytes — is specific to the hepatic asialoglycoprotein receptor subfamilies (SF470 ASGR1 and SF433 ASGR2, plus avian/non-mammalian hepatic lectin orthologs SF461 and SF454) and does not apply to myeloid mannose/PRR subfamilies (CD209, CLEC4E, CLEC6A) or IgE receptor FCER2.
ap2m1_cargo_selection	AP-2 complex subunit mu PANTHER:PTHR10529:SF236 Family	signal sequence receptor activity GO:0005048	Family Supported Family review supports GO:0005048 FAMILY_WIDE. Mu subunits across AP-1, AP-2, AP-3, and AP-4 directly bind peptide sorting signals (such as YxxPhi motifs) in the cytosolic tails of transmembrane cargos via a conserved hydrophobic pocket on the C-terminal MHD beta-sandwich.
cltc_coat_lattice	Clathrin heavy chain 1 PANTHER:PTHR10292:SF7 Family	structural molecule activity GO:0005198	Family Review Missing No supported structured family review is available for PANTHER:PTHR10292:SF7.
dnm2_vesicle_scission	Dynamin-2 PANTHER:PTHR11566:SF23 Family	GTPase motor activity GO:0061791	Family Review Missing No supported structured family review is available for PANTHER:PTHR11566:SF23.
atp6v1a_proton_pump	V-type proton ATPase catalytic subunit A PANTHER:PTHR43607:SF9 Family	proton-transporting ATPase activity, rotational mechanism GO:0046961	Family Review Missing No supported structured family review is available for PANTHER:PTHR43607:SF9.
ehd1_membrane_fission	EH domain-containing protein 1 PANTHER:PTHR11216:SF127 Family	ATP hydrolysis activity GO:0016887	Family Review Missing No supported structured family review is available for PANTHER:PTHR11216:SF127.
tsg101_escrt_sorting	Tumor susceptibility gene 101 protein PANTHER:PTHR23306:SF17 Family	ubiquitin binding GO:0043130	Family Review Missing No supported structured family review is available for PANTHER:PTHR23306:SF17.
rab7a_late_maturation	Ras-related protein Rab-7a PANTHER:PTHR47981:SF13 Family	GTPase activity GO:0003924	Family Review Missing No supported structured family review is available for PANTHER:PTHR47981:SF13.
ctsd_proteolysis	Cathepsin D PANTHER:PTHR47966:SF42 Family	aspartic-type endopeptidase activity GO:0004190	Family Review Missing No supported structured family review is available for PANTHER:PTHR47966:SF42.

Feedback loops (advisory, prose-driven: a regulator counts as "feedback" when its own description says so)

✓ every negative regulator described as feedback has an upstream edge closing the loop.

escrt_rab7_late_endosome ⊣ cytosolic_galnac_escape [closed]
escrt_rab7_late_endosome is regulated by early_endosome_acidification: feedback loop closed

Gene-review completeness (12/12 grounded genes reviewed)

8 complete review(s) · 6 with deep research · 0 missing review · 6 reviewed but lacking deep research

Gene	Review	Complete	Deep research
AP2M1 Q96CW1	✓	129/184	✗
ASGR1 P07306	✓	✓	✗
ASGR2 P07307	✓	✓	✗
ATP6V1A P38606	✓	✓	✓
CLTC Q00610	✓	✓	✓
CTSD P07339	✓	84/91	✗
DNM2 P50570	✓	170/171	✗
dpy-23 P35603	✓	22/23	✓
EHD1 Q9H4M9	✓	✓	✓
Rab7 P51150	✓	✓	✓
RAB7A P51149	✓	✓	✓
TSG101 Q99816	✓	✓	✗

Tree

Hepatocyte ASGR1/ASGR2 GalNAc-conjugate uptake, recycling, lysosomal routing, and cytosolic escapeTransport Stepasgr_galnac_hepatocyte_delivery

Parts
1. 1. sinusoidal hepatocyte ASGR1/ASGR2 recognition of GalNAc-conjugated payloads and desialylated glycoproteins
2. Cell-surface ASGR1/ASGR2 lectin binding of GalNAc conjugates & desialylated glycoproteinsMolecular Functionasgr_galnac_binding
  
  Annotons
  - ASGR1 (HL-1): major asialoglycoprotein receptor C-type lectin subunit Family: Asialoglycoprotein receptor 1
  - ASGR2 (HL-2): minor asialoglycoprotein receptor C-type lectin subunit Family: Asialoglycoprotein receptor 2
3. 2. clathrin/AP-2 coated-pit capture and DNM2-mediated vesicle scission
4. Clathrin/AP-2 coated pit assembly and DNM2 vesicle scissionTransport Stepclathrin_ap2_dnm2_internalization
  
  Annotons
  - AP2M1 (AP-2 mu2): tyrosine-motif cargo adaptor Family: AP-2 complex subunit mu
  - CLTC (CHC17): clathrin heavy chain coat lattice Family: Clathrin heavy chain 1
  - DNM2 (dynamin-2): coated-vesicle neck scission GTPase Family: Dynamin-2
5. 3. V-ATPase early endosome acidification and Ca2+-dependent ASGR-GalNAc uncoupling
6. V-ATPase early endosome acidification (pH drop & GalNAc ligand uncoupling from ASGR1/2)Transport Stepearly_endosome_acidification
  
  Annotons
  - ATP6V1A: V-ATPase catalytic subunit A Family: V-type proton ATPase catalytic subunit A
7. 4. EHD1-mediated tubular endocytic recycling of ligand-free ASGR1/ASGR2 to the hepatocyte surface
8. EHD1 recycling endosome tubulation and ASGR1/ASGR2 return to sinusoidal surfaceTransport Stepehd1_receptor_recycling
  
  Annotons
  - EHD1: recycling endosome tubulation/fission ATPase Family: EH domain-containing protein 1
9. 5. ESCRT-I MVB sorting and RAB7A-mediated late endosome maturation
10. TSG101/ESCRT-I MVB sorting and RAB7A late endosome maturation (degradative depot commitment)Transport Stepescrt_rab7_late_endosome
  
  Annotons
  - TSG101: ESCRT-I ubiquitin-binding MVB sorting subunit Family: Tumor susceptibility gene 101 protein
  - RAB7A / Rab7: late endosome maturation and HOPS-tethering GTPase Family: Ras-related protein Rab-7a
11. 6. rate-limiting endosomal membrane translocation and productive cytosolic GalNAc-oligonucleotide escape
12. Productive cytosolic GalNAc-siRNA/ASO escape (RISC / RNase H1 engagement)Transport Stepcytosolic_galnac_escape
13. 7. lysosomal fusion and CTSD-mediated luminal glycoprotein/conjugate catabolism
14. Lysosomal fusion and CTSD luminal cargo proteolysis/sequestrationMolecular Functionctsd_lysosomal_degradation
  
  Annotons
  - CTSD (cathepsin D): lysosomal aspartic endopeptidase Family: Cathepsin D

Details

Hepatocyte ASGR1/ASGR2 GalNAc-conjugate uptake, recycling, lysosomal routing, and cytosolic escapeTransport Stepasgr_galnac_hepatocyte_delivery

carbohydrate bindingGO:0030246 receptor-mediated endocytosisGO:0006898 endosomal lumen acidificationGO:0048388 endocytic recyclingGO:0032456 endosome to lysosome transportGO:0008333

Constructed for the DELIVEROME and ABM_TRAFFICKING projects to model hepatocyte-specific ASGR1/ASGR2 GalNAc-siRNA/ASO delivery and intracellular routing as an executable agent-based model (ABM) derived directly from the module graph. All protein participants are grounded to completed AIGR gene reviews (human ASGR1, ASGR2, AP2M1, CLTC, DNM2, ATP6V1A, EHD1, TSG101, RAB7A, CTSD, and mouse Rab7) and verified PANTHER family/subfamily identifiers.

Connections

asgr_galnac_binding -> clathrin_ap2_dnm2_internalization Provides Input For

GalNAc-bound ASGR1/ASGR2 complexes at the hepatocyte surface are recruited into clathrin/AP-2 coated pits and internalized via DNM2 scission.

clathrin_ap2_dnm2_internalization -> early_endosome_acidification Provides Input For

Internalized coated vesicles uncoat and fuse with V-ATPase-acidified early sorting endosomes where GalNAc ligands uncouple from ASGR1/ASGR2.

early_endosome_acidification -> ehd1_receptor_recycling Provides Input For

Ligand-free ASGR1/ASGR2 receptors partition into EHD1-dependent tubular recycling endosomes for rapid return to the sinusoidal plasma membrane.

early_endosome_acidification -> escrt_rab7_late_endosome Provides Input For

Uncoupled luminal GalNAc conjugates and desialylated glycoproteins progress via ESCRT-I (TSG101) and RAB7A into maturing late endosomes.

early_endosome_acidification -> cytosolic_galnac_escape Provides Input For

A fraction of internalized GalNAc oligonucleotide payload translocates across the limiting endosomal membrane into the hepatocyte cytosol.

escrt_rab7_late_endosome -> cytosolic_galnac_escape Negatively Regulates

Active RAB7A/ESCRT late endosomal maturation sequesters internalized payload into the late-endosomal/lysosomal pathway, limiting productive cytosolic escape (PMID:41814093).

escrt_rab7_late_endosome -> ctsd_lysosomal_degradation Provides Input For

Mature RAB7A-positive late endosomes fuse with lysosomes where CTSD degrades desialylated glycoproteins and sequesters un-escaped conjugates.

Part 1: sinusoidal hepatocyte ASGR1/ASGR2 recognition of GalNAc-conjugated payloads and desialylated glycoproteins

Cell-surface ASGR1/ASGR2 lectin binding of GalNAc conjugates & desialylated glycoproteinsMolecular Functionasgr_galnac_binding

Annotons

ASGR1 (HL-1): major asialoglycoprotein receptor C-type lectin subunit

asgr1_lectin_capture

Participant: Family: Asialoglycoprotein receptor 1

Family:

Asialoglycoprotein receptor 1

Representative Members: ASGR1 (human)UniProtKB:P07306

Function

asialoglycoprotein receptor activityGO:0004873

Substrates: trivalent GalNAc-siRNA/ASO conjugates and desialylated plasma glycoproteins

Processes

receptor-mediated endocytosisGO:0006898

Locations

external side of plasma membraneGO:0009897

Major H1 subunit of the hepatic Ashwell-Morell C-type lectin receptor that binds terminal Gal/GalNAc ligands via its QPD Ca2+-binding motif and presents its cytoplasmic YQDL tyrosine sorting motif to AP-2.

ASGR2 (HL-2): minor asialoglycoprotein receptor C-type lectin subunit

asgr2_lectin_capture

Participant: Family: Asialoglycoprotein receptor 2

Family:

Asialoglycoprotein receptor 2

Representative Members: ASGR2 (human)UniProtKB:P07307

Function

asialoglycoprotein receptor activityGO:0004873

Substrates: trivalent GalNAc-siRNA/ASO conjugates and desialylated plasma glycoproteins

Processes

receptor-mediated endocytosisGO:0006898

Locations

external side of plasma membraneGO:0009897

Minor H2 subunit that hetero-oligomerizes with ASGR1 on the sinusoidal hepatocyte surface to establish the multivalent CRD spacing required for nanomolar binding of triantennary GalNAc oligosaccharides and GalNAc-siRNA/ASO conjugates.

Part 2: clathrin/AP-2 coated-pit capture and DNM2-mediated vesicle scission

Clathrin/AP-2 coated pit assembly and DNM2 vesicle scissionTransport Stepclathrin_ap2_dnm2_internalization

Annotons

AP2M1 (AP-2 mu2): tyrosine-motif cargo adaptor

ap2m1_cargo_selection

Participant: Family: AP-2 complex subunit mu

Family:

AP-2 complex subunit muPANTHER:PTHR10529:SF236

Representative Members: AP2M1 (human)UniProtKB:Q96CW1 dpy-23 (C. elegans)UniProtKB:P35603

Function

signal sequence receptor activityGO:0005048

Processes

clathrin-dependent endocytosisGO:0072583 receptor internalizationGO:0031623

Locations

clathrin-coated pitGO:0005905

Recognizes the cytoplasmic YQDL tyrosine-based internalization motif of ASGR1 to concentrate GalNAc-bound receptor complexes into clathrin-coated pits.

CLTC (CHC17): clathrin heavy chain coat lattice

cltc_coat_lattice

Participant: Family: Clathrin heavy chain 1

Family:

Clathrin heavy chain 1PANTHER:PTHR10292:SF7

Representative Members: CLTC (human)UniProtKB:Q00610

Function

structural molecule activityGO:0005198

Processes

receptor-mediated endocytosisGO:0006898 clathrin coat assemblyGO:0048268

Locations

clathrin-coated endocytic vesicleGO:0045334

Assembles polyhedral clathrin cages around AP-2-clustered ASGR1/ASGR2 complexes to drive membrane invagination.

DNM2 (dynamin-2): coated-vesicle neck scission GTPase

dnm2_vesicle_scission

Participant: Family: Dynamin-2

Family:

Dynamin-2PANTHER:PTHR11566:SF23

Representative Members: DNM2 (human)UniProtKB:P50570

Function

GTPase motor activityGO:0061791

Processes

vesicle scissionGO:0099050 receptor-mediated endocytosisGO:0006898

Locations

clathrin-coated pitGO:0005905

Constricts and severs the neck of clathrin-coated pits to release ASGR-GalNAc vesicles into the hepatocyte cytoplasm.

Part 3: V-ATPase early endosome acidification and Ca2+-dependent ASGR-GalNAc uncoupling

V-ATPase early endosome acidification (pH drop & GalNAc ligand uncoupling from ASGR1/2)Transport Stepearly_endosome_acidification

Annotons

ATP6V1A: V-ATPase catalytic subunit A

atp6v1a_proton_pump

Participant: Family: V-type proton ATPase catalytic subunit A

Family:

V-type proton ATPase catalytic subunit APANTHER:PTHR43607:SF9

Representative Members: ATP6V1A (human)UniProtKB:P38606

Function

proton-transporting ATPase activity, rotational mechanismGO:0046961

Processes

endosomal lumen acidificationGO:0048388 proton transmembrane transportGO:1902600

Locations

endosome membraneGO:0010008 lysosomal membraneGO:0005765

Acidifies sorting endosomes to pH ~5.5-6.0, dissociating Ca2+ from the ASGR1/ASGR2 carbohydrate-recognition domains so that GalNAc conjugates are released into the endosomal lumen while free receptors enter recycling tubules.

Part 4: EHD1-mediated tubular endocytic recycling of ligand-free ASGR1/ASGR2 to the hepatocyte surface

EHD1 recycling endosome tubulation and ASGR1/ASGR2 return to sinusoidal surfaceTransport Stepehd1_receptor_recycling

Annotons

EHD1: recycling endosome tubulation/fission ATPase

ehd1_membrane_fission

Participant: Family: EH domain-containing protein 1

Family:

EH domain-containing protein 1PANTHER:PTHR11216:SF127

Representative Members: EHD1 (human)UniProtKB:Q9H4M9

Function

ATP hydrolysis activityGO:0016887

Processes

endocytic recyclingGO:0032456

Locations

recycling endosome membraneGO:0055038

Drives scission of recycling endosome tubules to rapidly return unoccupied ASGR1/ASGR2 receptors (~15-20 min cycle time) back to the hepatocyte sinusoidal membrane for repeated rounds of GalNAc uptake.

Part 5: ESCRT-I MVB sorting and RAB7A-mediated late endosome maturation

TSG101/ESCRT-I MVB sorting and RAB7A late endosome maturation (degradative depot commitment)Transport Stepescrt_rab7_late_endosome

Context

late endosome membraneGO:0031902

Annotons

TSG101: ESCRT-I ubiquitin-binding MVB sorting subunit

tsg101_escrt_sorting

Participant: Family: Tumor susceptibility gene 101 protein

Family:

Tumor susceptibility gene 101 proteinPANTHER:PTHR23306:SF17

Representative Members: TSG101 (human)UniProtKB:Q99816

Function

ubiquitin bindingGO:0043130

Processes

multivesicular body assemblyGO:0036258 ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathwayGO:0043162

Locations

late endosome membraneGO:0031902 early endosome membraneGO:0031901

Sorts ubiquitinated receptors and luminal cargo into multivesicular body intraluminal vesicles along the degradative trajectory.

RAB7A / Rab7: late endosome maturation and HOPS-tethering GTPase

rab7a_late_maturation

Participant: Family: Ras-related protein Rab-7a

Family:

Ras-related protein Rab-7aPANTHER:PTHR47981:SF13

Representative Members: RAB7A (human)UniProtKB:P51149 Rab7 (mouse)UniProtKB:P51150

Function

GTPase activityGO:0003924

Processes

early endosome to late endosome transportGO:0045022 endosome to lysosome transportGO:0008333

Locations

late endosomeGO:0005770 late endosome membraneGO:0031902

Drives maturation of sorting endosomes into late endosomes and promotes HOPS-dependent lysosomal fusion, sequestering GalNAc-conjugated cargo away from productive cytosolic escape.

Part 6: rate-limiting endosomal membrane translocation and productive cytosolic GalNAc-oligonucleotide escape

Productive cytosolic GalNAc-siRNA/ASO escape (RISC / RNase H1 engagement)Transport Stepcytosolic_galnac_escape

Context

cytosolGO:0005829 endosome membraneGO:0010008

PMID:29069408

Asialoglycoprotein receptor 1 mediates productive uptake of N-acetylgalactosamine-conjugated and unconjugated phosphorothioate antisense oligonucleotides into liver hepatocytes.

Only a minuscule fraction of endocytosed GalNAc-conjugated oligonucleotide escapes across endosomal membranes into the cytoplasm to mediate target cleavage, while the vast majority remains trapped in endolysosomal compartments.

PMID:41814093

In vivo endosomal escape assay identifies mechanisms for efficient hepatic LNP delivery.

Cross-modality hepatic nucleic-acid delivery study showing that Rab7-mediated late endosomal maturation competes with endosomal escape in hepatocytes and that loss of Rab7 enhances cytosolic delivery (used here as a qualitative mechanistic basis for the escrt_rab7_late_endosome -> cytosolic_galnac_escape inhibitory edge).

Loss of Rab7, a mediator of late endosomal maturation, increased LNP escape.

Part 7: lysosomal fusion and CTSD-mediated luminal glycoprotein/conjugate catabolism

Lysosomal fusion and CTSD luminal cargo proteolysis/sequestrationMolecular Functionctsd_lysosomal_degradation

Annotons

CTSD (cathepsin D): lysosomal aspartic endopeptidase

ctsd_proteolysis

Participant: Family: Cathepsin D

Family:

Cathepsin DPANTHER:PTHR47966:SF42

Representative Members: CTSD (human)UniProtKB:P07339

Function

aspartic-type endopeptidase activityGO:0004190

Processes

proteolysisGO:0006508

Locations

lysosomal lumenGO:0043202 endosome lumenGO:0031904

Degrades desialylated glycoproteins and represents the terminal lysosomal sink/depot for un-escaped GalNAc conjugates delivered via the RAB7A late-endosomal route.