The gene coding for HPSE consisting of 14 exons and 13 introns is located on chromosome 4q21.3 and expressed as two mRNAs (5 and 1.7 kb) by alternative splicing containing the same open reading frame (Dong et al., 2000). HPSE is initially synthesized as a preproenzyme of 68 kDa containing a signal sequence spanning Met1–Ala35, which is then processed into a proHPSE form after cleaving the signal sequence by signal peptidase. Lysosomal activation by cathepsin L excises a linker domain of Ser110–Gln157, giving rise to the mature HPSE as a non-covalent heterodimer containing an N-terminal 8 kDa (Gln36-Glu109) and a C-terminal 50 kDa (Lys158-Ile543) subunits.

β-glucuronidases are categorized into three GH families, GH1, GH2, and GH79, on the basis of their amino acid sequences (Henrissat and Davies, 1997; Cantarel et al., 2009). There are four characterized β-glucuronidase members in the GH79 family, including heparanase (EC 3.2.1.166), baicalin-β-D-glucuronidase (EC 3.2.1.167), 4-O-methyl-β-glucuronidase, and β-glucuronidase (Sasaki et al., 2000; Parish et al., 2001; Eudes et al., 2008; Konishi et al., 2008). Though both GH2 and GH79 belong to the GH-A clan, the GH79 family is composed of enzymes of both endo-acting HPSE and exo-acting β-glucuronidase, which contrasts that the GH2 family only consists of exo-acting β-glucuronidase. Folding prediction as well as multiple sequence alignment has predicted HPSE being a member of GH-A clan, proposing An (β/α)₈-TIM barrel as the key folding feature of HPSE (Nardella et al., 2004). This was confirmed after the recent determination of the long-anticipated hHPSE and hproHPSE structures.

The structure of apo hHPSE consists of a heterodimer formed by the 8-kDa subunit (residues Gln36–Glu109) and the 50-kDa subunit (Lys159–Ile543) (Wu et al., 2015), with the domain architecture comprising a catalytic (β/α)8-TIM barrel domain flanked by a β-sandwich domain (Figure 1B). Both the 8-kDa subunit and the 50-kDa subunit contribute to the formation of the catalytic (β/α)₈-TIM barrel and the β-sandwich domain. Though the β-sandwich domain was reported to facilitate secretion and activation, cellular trafficking, enzymatic and nonenzymatic activities of HPSE, its function demands to be further characterized (Simizu et al., 2007; Lai et al., 2008; Fux et al., 2009a). In addition, there are six putative N-glycosylation sites identified in the 50-kDa subunit of hHPSE. After the deglycosylation treatment of Endo-H during the protein preparation, N-linked GlcNAc residues were visible in the apo hHPSE structure at Asn162, Asn200, Asn217, Asn238 and Asn459, respectively (Wu et al., 2015). Intriguingly, glycosylation regulates HPSE secretion and endoplasmic reticulum-to-Golgi transport, but it is not required for enzymatic activity of HPSE (Simizu et al., 2004).

A binding groove of approximately 10 Å in the catalytic (β/α)₈-TIM barrel domain was recognized in the hHPSE structure. This binding groove contains residues Glu343 and Glu225, which are conserved in the GH79 family and have been previously identified as the catalytic nucleophile and acid-base pair of HPSE, suggesting that the HS-binding site is contained within this groove (Hulett et al., 2000). As shown in Figure 2A, the HPSE binding canyon is lined by side chains of basic residues, which correlates well with the negatively charged nature of HS substrates. Of interest is that the orientation of two subunits in the solved hHPSE structure implicates that the excised Ser110–Gln157 linker of the proHPSE could locate very close in space to the HS binding groove, which would physically clash the HS substrate. This is consistent with the reported hproHPSE structure in 2017, showing the restricted access to the active site cleft for oligosaccharide HS substrates due to the presence of the 6-kDa linker loop (Figure 2B) (Wu et al., 2017).

Structures of hHPSE in complex with HS analogues provides a structural rationale, clearly demonstrating that hHPSE recognizes a trisaccharide spanning the −2, −1 and +1 subsites, with the identical binding of GlcA at the −1 subsite in all bound HS analogues (Figure 2A). This conserved binding of GlcA is also observed in the GH79 bacterial members, suggesting a key GH79 structural motif that has been fine tuned to recognize GlcA (Michikawa et al., 2012; Wu et al., 2015). Significantly, N-sulfate at the −2 subsite and 6O-sulfate at the +1 subsite appear to be the main determinants for recognition of HS analogues due to their direct engagement in the hydrogen-bonding interactions with HPSE. 6O-sulfate at the −2 subsite and N-sulfate at the +1 subsite also contribute to the anchorage of HS substrates through electrostatic interactions to basic residues lining the active site cleft. Overall, structural information gained from the complexes of hHPSE with its HS substrate analogs is consistent with the findings of previous studies that HS sulfation patterns are essential for hHPSE enzymatic activity. Furthermore, sulfation contexts of HS substrates appear to act as a molecular signal that guides the precise cleavage of designated glycan sites. In addition, sulfate groups on the −2 and +1 moieties are implicated to aid hHPSE to unwind the substrate HS helix for a better access of the catalytic residues to facilitate the cleavage of the glycosidic bond.

When compared in the sequence alignment, a loop of 40 amino acids (Gly78–Thr117), which forms part of the exo-acting substrate-binding pocket identified in the AcaGH79 structure, corresponds to its counterpart that is substantially reduced in size of endo-acting BpHPSE (24 amino acids, Gly67–Pro90). Structurally, this shorter loop of BpHPSE allows the transition of the binding pocket into an open-end binding groove capable of accommodating elongated HS chains, which hereby provides a well-explained structure-based rationale of the discrepancy in acting modes of the substrate cleavage between the exo-AcaGH79 and endo-BpHPSE enzymes (Figure 2B).

The overall folding of hHPSE is comparable to that of two characterized bacterial GH79 members (Michikawa et al., 2012; Bohlmann et al., 2015), with Cα r.m.s. differences of 2.35 Å and 2.59 Å for AcaGH79 and BpHPSE, respectively. In particular, the 6 kDa linker peptide of hproHPSE that is proteolytically cleaved to enable the activation of hHPSE also corresponds to the AcaGH79 loop. Intriguingly, structural observations revealed that the physical presence of the hproHPSE linker peptide create a binding pocket on the protein surface containing those two highly conserved glutamate, resembling some structural characteristics of the exo-acting active site of AcGH79 (Wu et al., 2017). Detailed data indicate that this hproHPSE pocket is not involved in the HS interactions and GlcA occupation of the proHPSE pocket does not inhibit HPSE maturation, suggesting extra subsite interactions may be required for anchoring single GlcA molecules to proHPSE. Further investigations are thus required to determine whether this proHPSE pocket possesses any substrate specificity.

Whereas both hHPSE and BpHPSE demonstrate the endo-acting mode of substrate cleavage, differences exist between their substrates recognition. BpHPSE has preference for cleaving HS-containing GlcNAc residues (low sulfation), contrasting that GlcNS is preferably recognized by hHPSE. Moreover, sequence alignment of several eukaryotic HPSEs with BpHPSE and AcaGH79 revealed a remarkable conservation of key residues for accommodating GlcA at the −1 subsite of hHPSE, demonstrated by absolute conservation of residue Asn224, Glu225, Glu343, Gly350 and Tyr391 and evolutionary conservation of residue 62 (Asp of hHPSE vs Glu of BpHPSE and AcaGH79), 97 (Thr of hHPSE vs. Asn of BpHPSE and AcaGH79), 349 (Gly of hHPSE and BpHPSE vs Gln of AcaGH79), whilst residues at the −2 and +1 subsites show much poorer conservation in the BpHPSE and AcaGH79, thus providing a structure-based rationale for distinct substrate specificity amongst those GH79 enzymes (Wu et al., 2015).

It is generally known that mature HPSE is the only known endo-β-D-glucuronidase in mammals, which can cleave the HS chain of HSPG to release growth factors, chemokines, lipoproteins and enzymes, play a role in promoting tumors outside the cell. Many studies have shown that the function of HPSE is regulated by histones. Histones mainly exist in the nucleus and extracellular regions and secreted proteins. Histones (H1, H2A, H2B, H3, and H4) contain a large number of basic R-based amino acids, which are positively charged in aqueous solutions. While HSPG as one of the most negatively charged biopolymers, HSPG and histones bind to the GAG chain of HSPG through charge interaction to play a regulatory role. For example, extracellular histone H4 induces HS degradation by activating HPSE in chlorine (Cl2)-induced acute respiratory distress syndrome (ARDS). Knockdown of HPSE by RNAi demonstrated that histone h4-induced HS degradation requires HPSE and is dependent on the enzymatic activity of HPSE (Zhang et al., 2022). In cells expressing high levels of HPSE, reduction of nuclear syndecan-1 results in increased histone acetyltransferase (HAT) activity, which stimulates protein transcription and transcriptional upregulation of multiple genes that drive aggressive tumor phenotypes (Purushothaman et al., 2011). In lymphangiosarcoma (SS), histone deacetylase inhibitors (HDACi) upregulate HPSE by inducing the expression of the positive regulator EGR1 and inhibit the negative regulation of p53 by acetylation. By co-treatment with MEK inhibitor (trametinib) or HPSE inhibitor (SST0001/rooneparstat), blocking HDACi-induced erk-egr1-HPSE pathway enhanced antiproliferative and proapoptotic effects (Lanzi et al., 2021).

With the deepening of research, it was found that HPSE can inhibit tumor after entering the nucleus. Human HPSE sequences contain two potential nuclear localization signals (residues 271-277; PRRKTAK and residues 427-430; KRRK), which mediate nuclear localization of enzymes. Secondly, HPSE nuclear translocation can be promoted by its heparin binding domain, using HS as its carrier (Nadav et al., 2002). Yang et al. (2015) using atomic force microscopy and co-precipitation methods, found a direct molecular interaction between HPSE and DNA driven by charge, indicating that HPSE has dual functions in malignant melanoma, with primary extracellular activity and tumor-suppressive nuclear effect. In type 1 diabetes, heparin and HS can be transported to the nucleus and directly or indirectly affect gene transcription. Based on Chip-on-chip studies, heparin interacts with promoters and transcription regions of hundreds of genes and micro-RNAs in activated Jurkat T cells and upregulates transcription at the molecular level. Nuclear HPSE appears to regulate methylation of histone 3 lysine 4 (H3K4) by influencing demethylase recruitment of transcription-active genes (Parish et al., 2013).

As aforementioned, quite a few excellent reviews are available pertaining to activities of HPSE in different physiological and pathological contexts, we thus briefly summarize the pleiotropic actions of HPSE herein and will not go into detailed discussion (Figure 3). Function of HPSE is strongly associated with major human pathological complications, evidenced by that various literatures have linked overexpression of HPSE to enhanced tumor growth, metastasis and poor prognosis. Further, silencing of HPSE or treatment of tumor with compounds that block HPSE activity is shown to remarkably attenuate tumor progression. Therefore, targeting HPSE is considered as a promising therapeutic strategy for cancer treatment. Several classes of inhibitors have been developed, ranging from nucleic acid-based inhibitor, vaccines, MicroRNAs, anti-HPSE monoclonal antibodies, poly-sulfated saccharides to small-molecule inhibitors (Rivara et al., 2016). Though MicroRNAs and anti-HPSE antibodies are demonstrated to have high specificity, none of those so-called biological drugs, such as vaccines, antibodies, and antisense RNAs, have ever passed the clinical trials. Further, small molecule drugs also failed to enter clinical studies.

To the best of our knowledge, only few polysaccharide-based candidates synthesized by either semi-synthetic or total synthesis methods are currently clinical tested by competitively targeting the substrate binding site of HPSE. Irrespective of distinct mechanisms of action, those polysaccharide-based inhibitors, such as PI-88, M-402, PG545, and SST0001, appear to be the most promising anti-tumor agents due to their specificity and reasonable druggability. Significantly, the development of HPSE inhibitors still exist several drawbacks, among of which are structural uncertainty, per-sulfation, in vivo instability, poor bioavailability and apparent side effects (Rosenthal et al., 2002; Levidiotis et al., 2004; Hossain et al., 2010). As a result, novel strategies are emerging to develop HPSE inhibitors with higher specificity and greater selectivity (Sletten et al., 2017). Intriguingly, recent findings disclose that HPSE-2, a close homolog of HPSE but lacks enzymatic activity, can regulate antitumor mechanisms. However, this theme is not the main focus of this minireview, therefore it will not be further discussed.