ADAMTSL5 (ADAMTS-like protein 5; also known as THSD6) is a small secreted, N-glycosylated extracellular matrix glycoprotein of the ADAMTS superfamily. At 481 amino acids it is by far the smallest family member, and it is built from a signal peptide, a single thrombospondin type-1 (TSP1) repeat, a cysteine-rich module, a spacer module, and a C-terminal netrin-like (NTR) module joined to the spacer by a proline-rich disordered segment. Unlike the ADAMTS metalloproteinases it resembles, ADAMTSL5 has no metalloprotease or disintegrin-like domain and no proteolytic activity; it is a matrix-binding glycoprotein rather than an enzyme. The secreted protein binds heparin through its NTR module in a salt-sensitive, ionic interaction, and binds both fibrillin-1 and fibrillin-2, co-localising with fibrillin microfibrils in the extracellular matrix deposited by cultured fibroblasts; it is the first family member shown to bind both fibrillins. Its distribution is peri-cellular and baso-lateral, and part of the pool is proteolytically processed to release the C-terminal NTR-containing fragment. In mouse development the protein is broadly expressed, most prominently in musculoskeletal tissues including skeletal muscle, cartilage and bone, and in many epithelia. Whether ADAMTSL5 modulates microfibril biology is unresolved: adding it to fibroblast cultures changed neither microfibril density nor assembly. Beyond the matrix, a nine-residue peptide from its TSP1 repeat is presented by HLA-C*06:02 on melanocytes and acts as a CD8+ T cell autoantigen in psoriasis, and the gene is epigenetically activated in hepatocellular carcinoma, where it supports tumorigenicity in MET-sensitised hepatocytes.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0031012 extracellular matrix | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic propagation from PANTHER node PTN000347317, and an unusually well-founded one: all 16 protein donors in the WITH/FROM field carry their own experimental (IDA/HDA) annotation to this term or a descendant. Reason: Every WITH/FROM token was resolved and each donor's own evidence for the propagated term was queried. The GOA WITH/FROM field matches the cached PAINT seed list for this node exactly (17 tokens = 16 protein donors + the node id). All 16 protein donors hold their own IDA or HDA to GO:0031012 or a descendant, so any claim that the sources carry only family-level inference is contradicted by measurement. The target itself also holds an independent IDA to this term from PMID:23010571, and ADAMTSL5 is a signal-peptide secreted protein demonstrated to reside in fibroblast extracellular matrix. No specificity upgrade is warranted: the donor set is heterogeneous, spanning four distinct locations (extracellular matrix 14, basement membrane 4, interstitial matrix 1, microfibril 1), so GO:0031012 is the correct least common ancestor and refining it would mean arbitrarily preferring one donor's compartment. Separately checked and negative - the propagation does not land above its donors (14 of 16 hold this exact term), so no downward MODIFY applies. Propagation Review Root cause: NO FAILURE CORE Sources checked: FB:FBgn0003137 · Ppn (D. melanogaster) SUPPORTS TRANSFER Swiss-Prot Q868Z9; carries its own IDA to GO:0005604. FB:FBgn0032252 · loh (D. melanogaster) SUPPORTS TRANSFER TrEMBL Q9VKV3; carries its own IDA to GO:0031012. MGI:MGI:109249 · Adamts1 (M. musculus) SUPPORTS TRANSFER Swiss-Prot P97857; carries its own IDA to GO:0005604, GO:0031012. MGI:MGI:1347346 · Adamts7 (M. musculus) SUPPORTS TRANSFER Swiss-Prot Q68SA9; carries its own IDA to GO:0031012. MGI:MGI:1347356 · Adamts2 (M. musculus) SUPPORTS TRANSFER Swiss-Prot Q8C9W3; carries its own IDA to GO:0031012. MGI:MGI:1913798 · Adamtsl5 (M. musculus) SUPPORTS TRANSFER TrEMBL D3Z689; carries its own HDA/IDA to GO:0031012. MGI:MGI:1925044 · Adamtsl2 (M. musculus) SUPPORTS TRANSFER Swiss-Prot Q7TSK7; carries its own IDA to GO:0031012. MGI:MGI:2386139 · Papln (M. musculus) SUPPORTS TRANSFER Swiss-Prot Q9EPX2; carries its own HDA/IDA to GO:0005604, GO:0031012. MGI:MGI:2389008 · Adamtsl4 (M. musculus) SUPPORTS TRANSFER Swiss-Prot Q80T21; carries its own IDA to GO:0005614, GO:0031012. MGI:MGI:2672033 · Thsd4 (M. musculus) SUPPORTS TRANSFER Swiss-Prot Q3UTY6; carries its own IDA to GO:0001527, GO:0031012. PANTHER:PTN000347317 · PANTHER tree node (ADAMTS/ADAMTS-like family) SUPPORTS TRANSFER Internal PANTHER tree node, not a protein. The node carries four IBD terms but only GO:0031012 reached ADAMTSL5. RGD:621241 · Adamts1 (R. norvegicus) SUPPORTS TRANSFER Swiss-Prot Q9WUQ1; carries its own IDA to GO:0031012. UniProtKB:Q6ZMM2 · ADAMTSL5 (H. sapiens) - the target itself SUPPORTS TRANSFER Self-reference: the target is its own IBD seed, which is expected rather than circular -- its own IDA annotation to this term (PMID:23010571) is one of the descendant evidences behind the IBD. The IBA then asserts the additional claim that the function is inherited rather than lineage-specific. UniProtKB:Q8TE56 · ADAMTS17 (H. sapiens) SUPPORTS TRANSFER Swiss-Prot Q8TE56; carries its own IDA to GO:0031012. UniProtKB:Q9H324 · ADAMTS10 (H. sapiens) SUPPORTS TRANSFER Swiss-Prot Q9H324; carries its own IDA to GO:0031012. UniProtKB:Q9P2N4 · ADAMTS9 (H. sapiens) SUPPORTS TRANSFER Swiss-Prot Q9P2N4; carries its own IDA to GO:0031012. WB:WBGene00003242 · mig-6 (C. elegans) SUPPORTS TRANSFER Swiss-Prot O76840; carries its own IDA to GO:0005604. Supporting Evidence: PMID:23010571 co-localized with fibrillin microfibrils in the extracellular file:genes/human/ADAMTSL5/ADAMTSL5-bioinformatics/RESULTS.md All 16 protein donors carry their own experimental (IDA/HDA) annotation |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt Subcellular Location keyword mapping (SL-0243, Secreted). Correct and independently confirmed experimentally. Reason: ADAMTSL5 has a predicted signal peptide (residues 1-42) and a mature chain (43-481), and secretion was demonstrated directly: recombinant protein is recovered from the conditioned medium of three transfected cell lines. The SubCell-to-GO mapping is doing exactly what it should here, and the same term is independently supported by an IDA row on this gene. Supporting Evidence: PMID:23010571 Recombinant ADAMTSL5 is a secreted, N-glycosylated 60kDa |
| GO:0030198 extracellular matrix organization | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO transfer from IPR013273, a family signature that bundles the catalytic ADAMTS proteases together with the non-catalytic ADAMTS-like proteins. For the proteases, extracellular matrix organization follows from ECM proteolysis; ADAMTSL5 has no proteolytic activity, and the one direct test of matrix organization by ADAMTSL5 was negative. Reason: Two independent legs. First, the source signature is too broad to carry this process. InterPro:IPR013273 is named "ADAMTS/ADAMTS-like", covers 26,580 proteins, and carries exactly one GO mapping - this term. It matches ADAMTSL5 legitimately, but the ECM-organizing role of the family's catalytic members is a consequence of their metalloprotease activity, which ADAMTSL5 does not have: UniProt flags the missing catalytic domain in a CAUTION line, the primary paper states the ADAMTSLs have no proteolytic activity, and a sequence scan finds no HExxHxxGxxHD zinc-binding signature - indeed no HExxH substring at all - in the 481-residue sequence, with no peptidase signature among its InterPro matches. This is the familiar "a domain's name is not an activity" failure moved one aspect over, into a family signature too broad to carry a process. Second, the only direct experimental test was negative: adding ADAMTSL5 to fibroblast cultures produced no consistent difference in microfibril density and no discernible effect on microfibril assembly, and direct fibronectin binding was not supported either. Not REMOVE, deliberately: that negative is a "data not shown" result from a single exogenous-protein assay, so it shows no role has been demonstrated rather than refuting one, and UniProt itself only claims the protein may play a role in modulation of fibrillin microfibrils. A third argument was available and deliberately not used - ADAMTSL5 did not receive this term by IBA while ADAMTSL2, ADAMTSL4 and THSD4 did from the same node - because that node's propagation is incoherent family-wide (ADAMTSL1 and PAPLN received nothing at all), so the absence is more likely a coverage gap than a curatorial judgement. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE PSEUDO OR SUBACTIVITY LOSS Sources checked: InterPro:IPR013273 · ADAMTS/ADAMTS-like (family signature, 26580 proteins) SUPPORTS SOURCE BUT NOT TARGET Sole GO mapping is GO:0030198. The signature spans both catalytic ADAMTS proteases and non-catalytic ADAMTS-like proteins, so the mapping is sound for the protease members but does not transfer to a member that lost catalysis. Supporting Evidence: PMID:23010571 In contrast to ADAMTS proteases, ADAMTSLs lack a catalytic domain PMID:23010571 did not identify a consistent difference file:genes/human/ADAMTSL5/ADAMTSL5-uniprot.txt lacks the metalloprotease and disintegrin-like domains which are |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: Partner CYSRT1 (A8MQ03), from the HuRI yeast two-hybrid screen. An extreme Y2H hub (517 distinct IntAct partners) that localises to the cornified envelope, topologically inaccessible to a secreted ECM protein. Reason: Assessed per partner, not per gene. CYSRT1 has 517 distinct interaction partners in IntAct across 1670 records, against 12 for ADAMTSL5 - the signature of a promiscuous two-hybrid prey rather than a specific partner. Its only annotated location is the cornified envelope, an intracellular keratinocyte structure that a signal-peptide secreted protein (ADAMTSL5 signal peptide 1-42) never encounters. The interaction is also unreplicated: IntAct logs it under three sub-methods of the same experiment - two hybrid array, two hybrid prey pooling approach, and validated two hybrid - all at MI-score 0.56, so the NbExp=3 recorded in UniProt is one screen counted three ways, not three independent observations. No orthogonal assay exists. The accession resolves to the reviewed canonical Swiss-Prot entry at its canonical length (144 aa), so this is genuine screen noise rather than a mis-identified partner. Not REMOVE: the assay was performed and curated correctly by IntAct, it simply does not support a biologically meaningful binding function. Supporting Evidence: file:genes/human/ADAMTSL5/ADAMTSL5-bioinformatics/RESULTS.md CYSRT1 (A8MQ03) | 1670 | **517** | cornified envelope | 144 aa | |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: Partner KRTAP5-9 (P26371), from the same HuRI screen. A keratin-associated protein of the intracellular hair-keratin matrix, and a Y2H hub with 213 distinct partners. Reason: Assessed per partner. KRTAP5-9 is an ultrahigh-sulfur keratin-associated protein that assembles into the intracellular keratin matrix of the hair cuticle; it has no secretory pathway and cannot meet a secreted ECM glycoprotein in vivo. It carries 213 distinct IntAct partners across 842 records, again characteristic of a cysteine-rich, sticky two-hybrid prey. As with the other two rows the interaction is logged under three sub-methods of a single experiment at MI-score 0.56, so it is unreplicated. The accession is the reviewed canonical Swiss-Prot entry at its canonical length (169 aa). Not REMOVE, for the same reason as the CYSRT1 row: the annotation records a real screen result, but the bare term conveys no function and the partner is not credible. Supporting Evidence: file:genes/human/ADAMTSL5/ADAMTSL5-bioinformatics/RESULTS.md KRTAP5-9 (P26371) | 842 | **213** | intracellular hair-keratin matrix | 169 aa | |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: Partner FHL5 (Q5TD97), from the same HuRI screen. A nuclear LIM-domain transcriptional co-activator of spermatids, with 108 distinct IntAct partners. Reason: Assessed per partner. FHL5 is a four-and-a-half LIM domain protein annotated to the nucleus, specifically the nuclei of round and elongated spermatids, where it co-activates CREM. A secreted extracellular matrix glycoprotein and a nuclear transcriptional co-activator occupy mutually inaccessible compartments. FHL5 is also a substantial two-hybrid hub (108 distinct partners across 316 records), and like the other two partners it is supported only by three sub-methods of one experiment at MI-score 0.56 with no orthogonal validation. The accession is the reviewed canonical Swiss-Prot entry at its canonical length (284 aa). The three partners together cohere as a set of cysteine-rich, aggregation-prone Y2H preys rather than as any biology of ADAMTSL5. Supporting Evidence: file:genes/human/ADAMTSL5/ADAMTSL5-bioinformatics/RESULTS.md FHL5 (Q5TD97) | 316 | **108** | nucleus (spermatid nuclei) | 284 aa | |
| GO:0071953 elastic fiber | TAS PMID:23962539 Elastic fibres in health and disease. | MODIFY | Summary: Bulk assignment from a review article used as the TAS source for 62 distinct entities. The gene's own experimental evidence supports the more specific GO:0001527 microfibril, which UniProt already records by IDA but GOA is missing. Reason: PMID:23962539 is a review whose abstract never mentions ADAMTSL5, and QuickGO shows it is used as the reference for 66 annotations over 62 distinct entities, 41 of them receiving this same term - a bulk curation pass over the elastic fibre literature rather than a gene-specific traceable statement. Tellingly, the same curation from the same review assigned the more specific GO:0001527 microfibril to 15 other proteins including THSD4/ADAMTSL6, so the specific term was available and simply not chosen here. Meanwhile ADAMTSL5's own primary data demonstrates association with fibrillin microfibrils specifically, and UniProt already carries GO:0001527 microfibril by IDA for this protein - a term GOA does not have (QuickGO returns exactly 12 annotations for Q6ZMM2 and GO:0001527 is not among them). GO:0001527 is current, is not obsolete, and is a part_of child of GO:0071953, so replacing the review-derived parent with the evidence-backed child retains the parent by closure while gaining precision and aligns GOA with UniProt. The merged ADAMTSL4 review independently proposed GO:0001527 for its gene, which is useful convergent support. Note that elastic fibre is a poor fit on tissue grounds too: ADAMTSL5's strongest expression is in cartilage and bone, which are not elastic-fibre tissues. Proposed replacements: microfibril Supporting Evidence: PMID:23010571 co-localized with fibrillin microfibrils in the extracellular file:genes/human/ADAMTSL5/ADAMTSL5-uniprot.txt DR GO; GO:0001527; C:microfibril; IDA:UniProtKB. |
| GO:0050436 microfibril binding | IDA PMID:23010571 A disintegrin-like and metalloprotease domain containing thr... | ACCEPT | Summary: Core molecular function. Direct affinity co-isolation showed binding to both fibrillin-1 and fibrillin-2 and to assembled fibrillin microfibrils - ADAMTSL5 is the first family member shown to bind both fibrillins. Reason: Well supported by two complementary assays in the same paper: myc-tagged ADAMTSL5 co-isolated the N-terminal half of fibrillin-1 and both halves of fibrillin-2, with empty-vector controls showing no bead binding, and exogenous ADAMTSL5 added to fibroblast cultures decorated assembled fibrillin microfibrils. This is the most informative molecular function on the record and there is no more specific term available - GO has no fibrillin binding term (confirmed via OLS), and GO:0050436 is_a GO:0050840 extracellular matrix binding, so this is the appropriate level. Retained as a core function. Supporting Evidence: PMID:23010571 Taken together, the findings are consistent with specific binding of ADAMTSL5 to fibrillin-1 and fibrillin-2 and to their macromolecular assemblies, i.e., fibrillin microfibrils. |
| GO:0031012 extracellular matrix | IDA PMID:23010571 A disintegrin-like and metalloprotease domain containing thr... | ACCEPT | Summary: Direct demonstration that secreted ADAMTSL5 deposits into fibroblast extracellular matrix, with a non-permeabilised staining control establishing the signal is extracellular. MGI-assigned. Reason: The localisation was shown by immunostaining of fibroblast cultures exposed to ADAMTSL5, with two controls that make the call solid: empty-vector conditioned medium gave no signal, and staining without prior fixation reproduced the pattern, establishing that the decorated structures are extracellular rather than intracellular. UniProt records the same conclusion as an experimental subcellular location. One caveat recorded but not acted on: the assay used exogenous recombinant protein rather than protein at native levels, which by a strict reading leans toward IMP rather than IDA. I have not overturned the curator's call - for a secreted protein this is the standard assay and the non-permeabilised control addresses the main artefact. Supporting Evidence: PMID:23010571 suggesting that the stained structures were extracellular and corresponded to fibrillin-1 containing microfibrils in ECM |
| GO:0005576 extracellular region | IDA PMID:23010571 A disintegrin-like and metalloprotease domain containing thr... | ACCEPT | Summary: Secretion demonstrated directly - recombinant ADAMTSL5 is recovered from the conditioned medium of transfected HEK293F, COS-1 and CHO-K1 cells. MGI-assigned. Reason: Straightforward and correct, though it is the least informative of the three location rows since it is a parent of the extracellular matrix annotation the same paper supports. Secretion was reproduced in three independent cell lines and is consistent with the predicted signal peptide (1-42). Kept as non-redundant with GO:0031012 because it additionally captures the soluble medium pool, which matters for this protein: part of it is proteolytically processed to release the C-terminal NTR fragment into the medium. Supporting Evidence: PMID:23010571 in the medium of transfected cells |
| GO:0008201 heparin binding | IDA PMID:23010571 A disintegrin-like and metalloprotease domain containing thr... | ACCEPT | Summary: Core molecular function, mapped to a specific module. Heparin-agarose affinity chromatography showed salt-sensitive ionic binding, and only the C-terminal NTR-containing fragment was retained. Reason: Directly demonstrated and unusually well localised within the protein. ADAMTSL5 was depleted from medium by heparin-agarose, was not eluted by 0.15 or 0.3 M NaCl but was substantially eluted by 0.5 and 1 M NaCl, establishing an ionic interaction; and of the two fragments present in conditioned medium only the C-terminal NTR-containing one was retained, while the 33 kDa N-terminal fragment flowed through. This is consistent with the general heparin-binding property of NTR modules and gives the protein a plausible mechanism for its observed cell-surface and peri-cellular distribution via heparan sulfate proteoglycans. GO:0008201 is_a GO:0005539 glycosaminoglycan binding, which is the right level: the ligand assayed was heparin specifically. Retained as a core function. Supporting Evidence: PMID:23010571 only the C-terminal fragment containing the NTR-module was retained by the heparin matrix PMID:23010571 indicative of an ionic interaction between ADAMTSL5 and heparin |
| GO:0031012 extracellular matrix | IDA PMID:23010571 A disintegrin-like and metalloprotease domain containing thr... | ACCEPT | Summary: Duplicate of the MGI-assigned extracellular matrix IDA row, independently asserted by UniProt from the same paper. Same verdict. Reason: GOA carries this localisation twice, once assigned by MGI and once by UniProt, both citing PMID:23010571. Both are reviewed here separately so that every GOA row receives a verdict, and both are accepted on the same evidence. The duplication is a GOA bookkeeping artefact of two groups curating the same paper, not a second independent observation - worth noting so the row count is not read as two lines of support. The paper additionally localises the protein to the peri-cellular and baso-lateral matrix. Supporting Evidence: PMID:23010571 These observations strongly suggest that ADAMTSL5 is associated with the peri-cellular and sub-cellular ECM of cells |
| GO:0001527 microfibril | IDA PMID:23010571 A disintegrin-like and metalloprotease domain containing thr... | NEW | Summary: Proposed new annotation. This is the gene's most specific and best-evidenced localisation, it is already in UniProt by IDA, and GOA does not have it at all. Reason: GOA is missing the one localisation ADAMTSL5's own primary data establishes. UniProt's cross-reference block carries GO:0001527 C:microfibril IDA:UniProtKB, but QuickGO returns exactly 12 annotations for Q6ZMM2 and this term is not among them. The supporting experiment is direct: exogenous ADAMTSL5 added to fibroblast cultures decorated assembled fibrillin microfibrils, with an empty-vector control giving no signal and a non-permeabilised staining control establishing that the decorated structures are extracellular. Proposed as an additional row rather than relying solely on the MODIFY of the GO:0071953 TAS row, because that MODIFY alone would leave the specific term carrying the weak review-derived provenance of PMID:23962539, whereas the evidence that actually supports microfibril localisation is an IDA from PMID:23010571. Adding it here gives the UniProt/GOA discrepancy raised in suggested_questions a concrete target, and matches how the merged ADAMTSL4 review handled the same term. Supporting Evidence: PMID:23010571 the exogenous protein co-localized with fibrillin-1 in microfibrils file:genes/human/ADAMTSL5/ADAMTSL5-uniprot.txt DR GO; GO:0001527; C:microfibril; IDA:UniProtKB. |
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Download this section (compressed HTML)Q: PAINT recommendation, stated once for the whole family rather than per gene. Node PTN000347317 in PTHR13723 carries four IBD annotations, but their propagation to the eight human ADAMTSL/papilin members is incoherent: GO:0031012 reached 5 of 7 non-catalytic members (missing ADAMTSL1 and PAPLN) and GO:0030198 reached only 3 of 7 (missing ADAMTSL1, ADAMTSL3, ADAMTSL5 and PAPLN), while all four catalytic ADAMTS controls received all four terms. The sharpest case is human PAPLN, which received no IBA at all even though three of its own orthologs - Drosophila Ppn (FB:FBgn0003137), mouse Papln (MGI:MGI:2386139) and C. elegans mig-6/ppn-1 (WB:WBGene00003242) - are seeds for GO:0031012 at that very node. Human ADAMTSL1 likewise has no IBA at all despite being in the family (SF157). Is this a propagation-coverage gap rather than a set of deliberate curatorial exclusions, and can the node's terms be re-propagated across the clade?
Q: Related PAINT point, in the opposite direction. The family's loss of catalysis is modelled correctly - GO:0004222 and GO:0006508 reached all four catalytic ADAMTS controls and none of the seven ADAMTS-like members - but only ADAMTSL2 records this explicitly, via a NOT-qualified enables GO:0004222 IBA (IKR at node PTN002673039). For ADAMTSL1, ADAMTSL3, ADAMTSL4, ADAMTSL5, THSD4 and PAPLN the absence of catalytic activity is implicit, i.e. simply nothing propagated. Would placing the negation at the node ancestral to the whole non-catalytic clade make this informative loss explicit for all of them?
Q: Ontology issue. GO:0001527 microfibril is a part_of child of GO:0071953 elastic fiber, which asserts that every microfibril is part of an elastic fiber. This contradicts GO:0001527's own definition, which describes microfibrils as extracellular matrix components occurring independently or along with elastin, and contradicts the ciliary zonule - a fibrillin microfibril structure essentially devoid of elastin, and the reason FBN1 mutations cause ectopia lentis. Should the part_of be relaxed, or GO:0001527 re-parented under a fibrillin-microfibril concept independent of elastic fibre?
Q: UniProt/GOA discrepancy to reconcile. UniProt's cross-reference block carries GO:0001527 C:microfibril IDA:UniProtKB for ADAMTSL5, but GOA does not contain that annotation - QuickGO returns exactly 12 annotations for Q6ZMM2 and GO:0001527 is not among them. The gene's best-evidenced and most specific localisation is therefore missing from GOA, while a weaker review-derived TAS row to the broader parent GO:0071953 is present. Which record is stale?
Q: Does ADAMTSL5 have any demonstrable effect on fibrillin microfibril biology? The only test to date found no consistent difference in microfibril density and no discernible effect on assembly, in a single exogenous-protein assay reported as data not shown. Given that the protein binds both fibrillins and is proteolytically processed to release its NTR module, a loss-of-function experiment is needed before any extracellular matrix organization annotation is justified.
Suggested experts: Suneel S. Apte, Dieter P. Reinhardt
Q: What is the function, if any, of the released C-terminal NTR fragment? UniProt records that ADAMTSL5 is proteolytically cleaved to release an NTR-containing fragment, this fragment carries the heparin-binding activity, and it is detectable in mouse skeletal muscle, testis, kidney and heart. Is the cleavage regulated, and does the fragment have activity distinct from the full-length protein?
Suggested experts: Suneel S. Apte, Hannes L. Bader
Experiment: Generate Adamtsl5-null mice and examine fibrillin microfibril architecture in skeletal muscle, cartilage, bone and the ciliary zonule by immunofluorescence and transmission electron microscopy. This is the loss-of-function test the field lacks, and it would settle whether any extracellular matrix organization annotation is warranted. Compare against the Adamtsl2 and Thsd4 nulls, whose microfibril phenotypes are established.
Hypothesis: If ADAMTSL5 modulates fibrillin microfibril assembly in vivo, loss of the gene will perturb microfibril architecture in the tissues where the protein is most abundant.
Type: Mouse knockout with tissue ultrastructure
Experiment: Map the fibrillin-binding site. The primary paper showed ADAMTSL5 binds the N-terminal half of fibrillin-1 and both halves of fibrillin-2, with the N-terminal half binding more strongly. Narrow this with recombinant fibrillin subfragments and surface plasmon resonance to obtain an affinity and a domain-level site, then test competition with ADAMTSL2, ADAMTSL4 and THSD4.
Hypothesis: ADAMTSL5 binds a discrete N-terminal region shared by fibrillin-1 and fibrillin-2, and competes there with other microfibril-associated ADAMTSL proteins.
Type: Surface plasmon resonance with recombinant fragments
Experiment: Identify the protease that releases the NTR module and test whether the cleavage is regulated. Combine N-terminal sequencing or terminal-amine labelling of the released fragment with a protease-inhibitor panel in transfected cells, then ask whether the free NTR fragment and full-length ADAMTSL5 differ in heparin affinity and in microfibril decoration.
Hypothesis: Release of the C-terminal NTR module is a regulated processing event that changes the protein's matrix-binding behaviour.
Type: Protease identification and N-terminomics
Experiment: Compare ADAMTSL5 deposition on wild-type CHO cells with the glycosaminoglycan-deficient pgsA-745 and pgsD-677 mutants, and after heparinase III treatment. If the NTR-heparin interaction is what anchors the protein, retention should be lost in the mutants while secretion itself is unaffected.
Hypothesis: Peri-cellular retention of secreted ADAMTSL5 depends on the NTR-heparan sulfate interaction rather than on protein-protein contacts with the matrix.
Type: Cell-surface retention in glycosaminoglycan-deficient mutants
Experiment: Test whether purified recombinant ADAMTSL5 added to the medium reproduces the changes in MET, EGFR and FGFR4 seen on knockdown, and whether the effect requires the NTR module. This would distinguish a bona fide extracellular signal from an intracellular or indirect consequence of depleting the gene, and would determine whether any signalling GO annotation is ever justified.
Hypothesis: The reported effect of ADAMTSL5 on receptor tyrosine kinase levels in hepatocellular carcinoma is a genuine extracellular, matricrine activity requiring the heparin-binding NTR module.
Type: Recombinant protein add-back with domain deletion
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