1.1 Overview of the hyaluronidases
The hyaluronidases (Hyals) are classes of enzymes that degrade predominantly hyaluronan (HA). The term “hyaluronidase” is somewhat of a misnomer since they have the limited ability to degrade chondroitin (Ch) and chondroitin sulfates (ChS), albeit at a slower rate. It is a common misconception that the bacterial Hyals have absolute specificity for HA. This is incorrect. Both bacterial 1 and vertebrate enzymes degrade Ch and ChS, albeit at a slower rate. The plausible reason for this broader specificity is that chondroitins preceded HA in evolution. For example, the nematode, Caenorhabditis elegans, contains only Ch and no HA, with only one Hyal-like sequence (unpublished observations). This is most likely a chondroitinase. It is plausible, therefore, that the vertebrate Hyals evolved originally from pre-existing chondroitinases 1. This may explain why Hyals, recognizing their ancestral substrate, retain limited ability to also degrade Ch and ChS.

The Hyals from bacteria have been well characterized, and much information is available (for representative publications see 2–5). The Hyals in vertebrate tissues, on the other-hand, have not been studied extensively, however, due to the lack of structural information. Such studies were more difficult and, therefore, more limited. In addition, vertebrate Hyals are present at exceedingly low concentrations. In human serum, e.g., Hyal1 is present at 60 ng/ml 6. They have high specific activities that are unstable during the course of purification, requiring the constant presence of detergents and protease inhibitors for their isolation. Many of such difficulties have been overcome, and a great deal of information is now available, facilitated in part by the Human Genome Project 7.

Six Hyal sequences occur in the human genome, constituting a newly recognized family of enzymes. They have similar catalytic mechanisms that contrast markedly with the bacterial Hyals. There is growing interest in these enzymes as their HA substrate is achieving much attention. An outstanding review of the hyaluronidases was published 50 years ago by Karl Meyer, who was also the first to describe the chemical structure of HA 8. Interestingly, a chapter on mucopolysaccharidases, the former name for the hyaluronidases, was included in Volume 1 of Methods in Enzymology 9. The most recent overview of all of the Hyals appeared in 1971 10. Since that time, no comprehensive review has appeared.

Karl Meyer classified the Hyals into three distinct classes of enzymes 8, based entirely on the biochemical analyses available at the time. With the advent of sequence and structural data, we can now appreciate how remarkably accurate Karl Meyer’s classification scheme was. No modification of his formulation is necessary. There are three major groups of Hyals, based on their mechanisms of action. Two of the groups are endo-β-N-acetyl-hexosaminidases. One group includes the vertebrate enzymes that utilize substrate hydrolysis 11,12. The second group, which is predominantly bacterial, includes the eliminases that function by β-elimination of the glycosidic linkage with introduction of an unsaturated bond 2–4,13–17. As these enzymes catalyze the breaking of chemical bond by means other than hydrolysis or oxidation, and with the forming a new double bond they are also termed lyases. Both terms, the eliminase (or β-eliminase) and the lyase, are used in the review interchangeably. The third group are the endo-β-glucuronidases. These are found in leeches, which are annelids 18, and in certain crustaceans 19. No sequence data are available, and little is known about this potentially interesting class of enzymes. However, their mechanism of action resembles that of the eukaryotic or vertebrate enzymes more closely than the bacterial enzymes.

Sequence data for vertebrate Hyals now provide opportunities to formulate structure-function relationships, to examine probable mechanisms of catalysis, to identify putative substrate binding sites, and to consider the additional non-enzymatic functions of this family of multifunctional enzymes for two of the three groups, for the hydrolase and lyase types of Hyals, respectively 2. Such a review is presented here, documenting some of the common and some of the unusual features that distinguish each of these families of enzymes.

The primary objective of this review is to clarify what is known about the structure and mode of action of all the Hyals. Since so little is known of the leech-type of Hyals, the β-endoglucuronidases, the emphasis will, by necessity, be upon two of the three classes of enzymes. Other aspects of these enzymes, such as their physiological activities, their dependence on reaction conditions, their role in cell biology and involvement in metabolism, and their use as reagents or as therapeutics, are not the concern presently. A review of these other aspects of the Hyals will appear separately (Stern and Jedrzejas, in preparation).

High levels of HA turnover occur in vertebrate tissues. Tight regulation of catabolism is crucial for modulating steady state levels, important for normal homeostasis, and for embryonic development, wound healing, regeneration, and repair. Under pathological conditions, as in severe stress, shock, septicemia, in burn patients, following major surgery, massive injury, circulating HA levels increases rapidly. HA also increases in association with aggressive malignancies. Determining the mechanism of action of the Hyals is critical for understanding their controls over such a wide range of functions (for reviews, see 20,21).

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Hyaluronidases: their genomics, structures, and mechanisms of action.

Insect pests and pathogens (fungi, bacteria and viruses) are responsible for severe crop losses. Insects feed directly on the plant tissues, while the pathogens lead to damage or death of the plant. Plants have evolved a certain degree of resistance through the production of defence compounds, which may be aproteic, e.g. antibiotics, alkaloids, terpenes, cyanogenic glucosides or proteic, e.g. chitinases, β-1,3-glucanases, lectins, arcelins, vicilins, systemins and enzyme inhibitors. The enzyme inhibitors impede digestion through their action on insect gut digestive α-amylases and proteinases, which play a key role in the digestion of plant starch and proteins. The natural defences of crop plants may be improved through the use of transgenic technology. Current research in the area focuses particularly on weevils as these are highly dependent on starch for their energy supply. Six different α-amylase inhibitor classes, lectin-like, knottin-like, cereal-type, Kunitz-like, γ-purothionin-like and thaumatin-like could be used in pest control. These classes of inhibitors show remarkable structural variety leading to different modes of inhibition and different specificity profiles against diverse α-amylases. Specificity of inhibition is an important issue as the introduced inhibitor must not adversely affect the plant's own α-amylases, nor the nutritional value of the crop. Of particular interest are some bifunctional inhibitors with additional favourable properties, such as proteinase inhibitory activity or chitinase activity. The area has benefited from the recent determination of many structures of α-amylases, inhibitors and complexes. These structures highlight the remarkable variety in structural modes of α-amylase inhibition. The continuing discovery of new classes of α-amylase inhibitor ensures that exciting discoveries remain to be made. In this review, we summarize existing knowledge of insect α-amylases, plant α-amylase inhibitors and their interaction. Positive results recently obtained for transgenic plants and future prospects in the area are reviewed.

Plant alpha-amylase inhibitors and their interaction with insect alpha-amylases.

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

/pdf/in-posidonia-oceanica-cadmium-induces-changes-in-dna-3cu7uj83mr.pdf

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

There is emerging evidence that the proteolytic machinery of plants plays important roles in defense against pathogens. The oomycete pathogen Phytophthora infestans, the agent of the devastating late blight disease of tomato (Lycopersicon esculentum) and potato (Solanum tuberosum), has evolved an arsenal of protease inhibitors to overcome the action of host proteases. Previously, we described a family of 14 Kazal-like extracellular serine protease inhibitors from P. infestans. Among these, EPI1 and EPI10 bind and inhibit the pathogenesis-related (PR) P69B subtilisin-like serine protease of tomato. Here, we describe EPIC1 to EPIC4, a new family of P. infestans secreted proteins with similarity to cystatin-like protease inhibitor domains. Among these, the epiC1 and epiC2 genes lacked orthologs in Phytophthora sojae and Phytophthora ramorum, were relatively fast-evolving within P. infestans, and were up-regulated during infection of tomato, suggesting a role during P. infestans-host interactions. Biochemical functional analyses revealed that EPIC2B interacts with and inhibits a novel papain-like extracellular cysteine protease, termed Phytophthora Inhibited Protease 1 (PIP1). Characterization of PIP1 revealed that it is a PR protein closely related to Rcr3, a tomato apoplastic cysteine protease that functions in fungal resistance. Altogether, this and earlier studies suggest that interplay between host proteases of diverse catalytic families and pathogen inhibitors is a general defense-counterdefense process in plant-pathogen interactions.

A Phytophthora infestans Cystatin-Like Protein Targets a Novel Tomato Papain-Like Apoplastic Protease

Elevated CO2 and temperature are altering the interactions between plants and insects with important implications for food security and natural ecosystems. Ecologically, the acceleration of plant phenology by warming is generating mismatches between plants and insect pollinators. Similarly, shifting

/pdf/climate-change-resetting-plant-insect-interactions-r0xbf7ednw.pdf

Climate change: Resetting plant-insect interactions

The spread, classification, and properties of plant proteins capable of inhibiting proteinases have been reviewed. Data from the literature on the likely physiological functions of these inhibitors in plants are analyzed.

[Proteinase inhibitors and their function in plants: a review].

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2898%2900321-4

Kunitz-type proteinase inhibitors from intact and Phytophthora-infected potato tubers

The recently described inhibitor of cysteine proteinases from Trypanosoma cruzi, chagasin, was found to have close homologs in several eukaryotes, bacteria and archaea, the first protein inhibitors of cysteine proteases in prokaryotes. These previously uncharacterized 110–130 residue-long proteins share a well-conserved sequence motif that corresponds to two adjacent β-strands and the short loop connecting them. Chagasin-like proteins also have other conserved, mostly aromatic, residues, and share the same predicted secondary structure. These proteins adopt an all-β fold with eight predicted β-strands of the immunoglobulin type. The phylogenetic distribution of the chagasins generally correlates with the presence of papain-like cysteine proteases. Previous studies have uncovered similar trends in cysteine proteinase binding by two unrelated inhibitors, stefin and p41, that belong to the cystatin and thyroglobulin families, respectively. A hypothetical model of chagasin–cruzipain interaction suggests that chagasin may dock to the cruzipain active site in a similar manner with the conserved NPTTG motif of chagasin forming a loop that is similar to the wedge structures formed at the active sites of papain and cathepsin L by stefin and p41.

Sequence conservation in the chagasin family suggests a common trend in cysteine proteinase binding by unrelated protein inhibitors

Possible utilities for natural inhibitors of proteolytic enzymes in plant biotechnology have been reviewed. Among the potential areas of use of the inhibitors are (1) construction of transgenic plants with increased resistance to insects and other pests and (2) development of procedures for biosynthesis of recombinant proteins. In the latter case, the inhibitors will serve to prevent the protein degradation by proteinases.

[Proteinase inhibitors in plant biotechnology: a review].

The serine proteinase inhibitor (PSPI-21) isolated from potato tubers (Solanum tuberosum L.) comprises two protein species with pI 5.2 and 6.3, denoted as PSPI-21-5.2 and PSPI-21-6.3, respectively. They were separated by anion exchange chromatography on a Mono Q FPLC column. Both species tightly inhibit human leukocyte elastase, whereas their interaction with trypsin and chymotrypsin is substantially weaker. The sequences of both PSPI-21-5.2 and PSPI-21-6.3 were determined by analysis of overlapping peptides obtained from the oxidized or reduced and S-pyridylethylated proteins after digestion with trypsin or pepsin. Both species of PSPI-21 are composed of two chains, named chains A and B, which are linked by a disulfide bridge between Cys(146) and Cys(157). The other disulfide bridge is located within the A chains between Cys(48) and Cys(97). The amino acid sequences of the large A chains of the two forms, consisting of 150 amino acids residues each, differ in a single residue at position 52. The small chains B, containing 37 and 36 residues in PSPI-21-6.3 and PSPI-21-5.2, respectively, have nine different residues. The entire amino acid sequences of the two inhibitors show a high degree of homology to the other Kunitz-type proteinase inhibitors from plants.

/pdf/primary-structure-of-potato-kunitz-type-serine-proteinase-3pfgmmq2zw.pdf

V. V. Mosolov

Papers

[Proteinase inhibitors and their function in plants: a review].

Kunitz-type proteinase inhibitors from intact and Phytophthora-infected potato tubers

Sequence conservation in the chagasin family suggests a common trend in cysteine proteinase binding by unrelated protein inhibitors

[Proteinase inhibitors in plant biotechnology: a review].

Primary structure of potato kunitz-type serine proteinase inhibitor.