Expressing the cloned naked mole rat HAS2 cDNA in human HEK293 cells is sufficient to make those cells secrete high-molecular-mass hyaluronan. This is a direct assay of the activity of the naked mole rat protein in a heterologous host, i.e. IDA-grade evidence for hyaluronan synthase activity on G5AY81 itself rather than an ortholog projection.

Knocking down HAS2 by stable shRNA in naked mole rat fibroblasts abolishes high-molecular-mass hyaluronan production, giving loss-of-function (IMP-grade) support for the hyaluronan biosynthetic process annotation in this species.

The naked mole rat HAS2 protein carries two substitutions of asparagines that are invariant across mammals to serine, located in the cytoplasmic loop that contains the active site.

Naked mole rat skin fibroblasts express HAS2 at higher levels than mouse or human fibroblasts, whereas HAS1 and HAS3 levels are comparable.

Hyaluronan produced by HAS2 is causally responsible for the naked mole rat's elevated resistance to malignant transformation and tumour formation.

Transgenic expression of the naked mole rat Has2 gene in mice raises hyaluronan levels in multiple tissues and lowers cancer incidence while extending lifespan and healthspan - an in vivo gain-of-function complement to the HEK293 experiment.

Hyaluronan from nmrHas2 mouse tissues is both more abundant and of higher molecular mass, confirming that the naked mole rat enzyme raises polymer mass and not only polymer amount.

Among the three HAS isoenzymes, Has2 is the one that makes high-molecular-mass hyaluronan, and it is more highly expressed in naked mole rats than in mice or humans.

The naked mole rat's high-molecular-mass hyaluronan phenotype is postnatal, which is consistent with the embryonic/developmental behaviour of the enzyme being the conserved, not the divergent, part of its biology.

HAS2 is the long-polymer synthase of the family - a general mammalian property, not a naked mole rat peculiarity.

Naked mole rat and Damaraland mole rat cells express HAS2 at dramatically higher levels than their aboveground comparators.

In a subterranean sister species (blind mole rat), HAS2 coding sequence alone, at matched expression, yields larger hyaluronan than the mouse enzyme - direct evidence that HAS2 sequence changes can set polymer length independently of expression level.

One of the two naked mole rat HAS2 substitutions is shared by all African mole-rats rather than being private to the naked mole rat.

HAS2 is under strong purifying selection across mammals, which argues that the conserved ancestral functions of the enzyme are retained in the naked mole rat and that its divergence is a refinement rather than a replacement.

HAS enzymes polymerise hyaluronan at the inner face of the plasma membrane and extrude it directly into the extracellular matrix - the basis for treating the plasma membrane as the functional location and extracellular matrix assembly as a direct process.

Independent re-measurement by size-exclusion chromatography and gel electrophoresis finds naked mole rat hyaluronan larger and more abundant than the guinea pig comparator, but with a maximum near 2.5 MDa rather than 6-12 MDa.

The renal medulla is the single tissue in which naked mole rat hyaluronan content runs against the species trend, being lower than in the comparator species.

Naked mole rat renal medullary hyaluronan content resembles that of desert gerbils, a lineage in which low medullary hyaluronan accompanies maximal water conservation.

The rat and mouse renal phenotype that grounds the transferred renal GO terms is a water-loading-responsive increase in medullary interstitial hyaluronan that reduces water reabsorption; the corresponding experiment has never been done in the naked mole rat.

The authors state explicitly that the naked mole rat response to water loading is unknown, so the physiological role of medullary hyaluronan in this species is untested.

The naked mole rat's urine-concentrating ability is described as only moderate, not exceptional.

The community consensus upholds the qualitative claim that naked mole rat hyaluronan is larger than that of comparator mammals and has unusual material properties, while noting that reported sizes vary with isolation procedure.

The quantitative 6-12 MDa figure is not upheld; the best available re-measurement puts the average molecular weight below 2.5 MDa in both tissue and cell supernatant.

The mechanistic link from the two Asn-to-Ser substitutions to increased polymer length is not established, since heterologous expression of naked mole rat HAS2 produced a range of hyaluronan sizes depending on the host cell.

The cytoprotective effect of hyaluronan scales with polymer length, establishing that polymer mass, not merely hyaluronan presence, is the biologically decisive variable - the justification for proposing a polymer-length-specific biosynthesis term.

Naked mole rat hyaluronan forms tissue-specific folded assemblies and spontaneously gels without chemical cross-linking, an extracellular-matrix-forming behaviour not seen with commercial hyaluronan.

High-molecular-weight hyaluronan accumulating in naked mole rat tissues is credited with the species' resistance to post-traumatic osteoarthritis, a further downstream consequence of Has2 output.

Naked mole rat TMEM2 is not a physiological hyaluronidase, showing that the tissue accumulation of hyaluronan in this species is partly a catabolic defect and not attributable to Has2 alone.

The naked mole rat hyaluronan phenotype is jointly attributable to increased synthase processing/production capacity and reduced hyaluronidase-mediated degradation.