Acid Endopeptidase Research Articles

Aspergillus oryzae PrtR alters transcription of individual peptidase genes in response to the growth environment

Aspergillus oryzae PrtR is an ortholog of the transcription factor PrtT, which positively regulates the transcription of extracellular peptidase genes in Aspergillus niger and Aspergillus fumigatus. To identify the genes under the control of PrtR and elucidate its regulatory mechanism in A. oryzae, prtR gene disruption mutants were generated. The control strain clearly showed a halo on media containing skim milk as the nitrogen source, whereas the ΔprtR strain formed a smaller halo. Measurement of acid peptidase activity revealed that approximately 84% of acidic endopeptidase and 86% of carboxypeptidase activities are positively regulated by PrtR. As the transcription of the prtR gene varied depending on culture conditions, especially with or without a protein substrate, it was considered that its transcription would be regulated in response to a nitrogen source. In addition, contrary to previous expectations, PrtR was found to act both in promoting and repressing the transcription of extracellular peptidase genes. The mode of regulation varied from gene to gene. Some genes were regulated in the same manner in both liquid and solid cultures, whereas others were regulated in different ways depending on the culture conditions. Furthermore, PrtR has been suggested to regulate the transcription of peptidase genes that are closely associated with other transcription factors.Key points• Almost all peptidase genes in Aspergillus oryzae are positively regulated by PrtR• However, several genes are regulated negatively by PrtR• PrtR optimizes transcription of peptidase genes in response to culture conditions

AoS28D, a proline-Xaa carboxypeptidase secreted by Aspergillus oryzae.

Prolyl peptidases of the MEROPS S28 family are of particular interest because they are key enzymes in the digestion of proline-rich peptides. A BLAST analysis of the Aspergillus oryzae genome revealed sequences coding for four proteases of the S28 family. Three of these proteases, AoS28A, AoS28B, and AoS28C, were previously characterized as acidic prolyl endopeptidases. The fourth protease, AoS28D, showed high sequence divergence with other S28 proteases and belongs to a phylogenetically distinct cluster together with orthologous proteases from other Aspergillus species. The objective of the present paper was to characterize AoS28D protease in terms of substrate specificity and activity. AoS28D produced by gene overexpression in A. oryzae and in Pichia pastoris was a 70-kDa glycoprotein with a 10-kDa sugar moiety. In contrast with other S28 proteases, AoS28D did not hydrolyze internal Pro-Xaa bonds of several tested peptides. Similarly, to human lysosomal Pro-Xaa carboxypeptidase, AoS28D demonstrated selectivity for cleaving C-terminal Pro-Xaa bonds which are resistant to carboxypeptidases of the S10 family concomitantly secreted by A. oryzae. Therefore, AoS28D could act in synergy with these enzymes during sequential degradation of a peptide from its C-terminus.

Major acid endopeptidases of the blood-feeding monogenean Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae).

In parasitic flatworms, acid endopeptidases are involved in crucial processes, including digestion, invasion, interactions with the host immune system, etc. In haematophagous monogeneans, however, no solid information has been available about the occurrence of these enzymes. Here we aimed to identify major cysteine and aspartic endopeptidase activities in Eudiplozoon nipponicum, an invasive haematophagous parasite of common carp. Employing biochemical, proteomic and molecular tools, we found that cysteine peptidase activities prevailed in soluble protein extracts and excretory/secretory products (ESP) of E. nipponicum; the major part was cathepsin L-like in nature supplemented with cathepsin B-like activity. Significant activity of the aspartic cathepsin D also occurred in soluble protein extracts. The degradation of haemoglobin in the presence of ESP and worm protein extracts was completely inhibited by a combination of cysteine and aspartic peptidase inhibitors, and diminished by particular cathepsin L, B and D inhibitors. Mass spectrometry revealed several tryptic peptides in ESP matching to two translated sequences of cathepsin L genes, which were amplified from cDNA of E. nipponicum and bioinformatically annotated. The dominance of cysteine peptidases of cathepsin L type in E. nipponicum resembles the situation in, e.g. fasciolid trematodes.

House dust mites possess a polymorphic, single domain putative peptidoglycan d,l endopeptidase belonging to the NlpC/P60 Superfamily

A 14kDa protein homologous to the γ-d-glutamyl-l-diamino acid endopeptidase members of the NlpC/P60 Superfamily has been described in Dermatophagoides pteronyssinus and Dermatophagoides farinae but it is not clear whether other species produce homologues. Bioinformatics revealed homologous genes in other Sarcopteformes mite species (Psoroptes ovis and Blomia tropicalis) but not in Tetranychus urticae and Metaseiulus occidentalis. The degrees of identity (similarity) between the D. pteronyssinus mature protein and those from D. farinae, P. ovis and B. tropicalis were 82% (96%), 77% (93%) and 61% (82%), respectively. Phylogenetic studies showed the mite proteins were monophyletic and shared a common ancestor with both actinomycetes and ascomycetes. The gene encoding the D. pteronyssinus protein was polymorphic and intronless in contrast to that reported for D. farinae. Homology studies suggest that the mite, ascomycete and actinomycete proteins are involved in the catalysis of stem peptide attached to peptidoglycan. The finding of a gene encoding a P60 family member in the D. pteronyssinus genome together with the presence of a bacterial promotor suggests an evolutionary link to one or more prokaryotic endosymbionts.

Structures of a Bifunctional Cell Wall Hydrolase CwlT Containing a Novel Bacterial Lysozyme and an NlpC/P60 dl-Endopeptidase

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).

The crystal structure of an intermediate dimer of aspergilloglutamic peptidase that mimics the enzyme-activation product complex produced upon autoproteolysis

Aspergilloglutamic peptidase from Aspergillus niger var. macrosporus (AGP) is one of the so-called pepstatin-insensitive acid endopeptidases, which are distinct from the well-studied aspartic peptidases. Among the known homologues of the glutamic peptidases, AGP is a unique two-chain enzyme with a light chain and a heavy chain bound non-covalently with each other, and thus is an interesting target for protein structure-function relationship studies. In this article, we report the crystal structure of a dimeric form of the enzyme at a resolution of 1.6 Å. This form has a unique structure in which the C-terminal region of the light chain of one of the molecules binds to the active site cleft of the other molecule like a part of a substrate. This form mimics the enzyme-activation product complex produced upon autoproteolysis, and provides a structural clue that could help to clarify the activation mechanism. This type of dimeric structure of a peptidase is here reported for the first time.

Primary structure of turkey myoglobin

Our objective was to determine the amino acid sequence of turkey myoglobin. Turkey myoglobin was isolated from cardiac muscles via ammonium sulphate precipitation and gel-filtration chromatography. Purified turkey myoglobin, separated as a 17 kDa band in SDS–PAGE, was subjected to digestion with trypsin or aspartic acid endopeptidase. The resulting peptides were separated by reverse-phase HPLC, and then subjected to Edman degradation to obtain the amino acid sequence. The complete amino acid sequence of turkey myoglobin was determined and compared with that of poultry and red meat myoglobins. Turkey myoglobin has 153 amino acids and nine histidine residues. Proximal (position 93) and distal (position 64) histidine residues, responsible for maintaining the stability of haeme, are conserved in turkey myoglobin. Turkey myoglobin shares 100% sequence similarity with chicken myoglobin, whereas it shares 92.5% homology with ostrich, 76.5% with pig, and less than 73% with ruminant myoglobins.

Structure of the γ-D-glutamyl-L-diamino acid endopeptidase YkfC from Bacillus cereus in complex with L-Ala-γ-D-Glu: insights into substrate recognition by NlpC/P60 cysteine peptidases.

Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-D-glutamyl-L-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (L-Ala-γ-D-Glu) enabled the identification of conserved sequence and structural signatures for recognition of L-Ala and γ-D-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial L-alanine-γ-D-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site.

Structural Basis of Murein Peptide Specificity of a γ-D-Glutamyl-L-Diamino Acid Endopeptidase

The crystal structures of two homologous endopeptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme were determined at 1.05 and 1.60 A resolution, respectively, and contain a bacterial SH3-like domain (SH3b) and a ubiquitous cell-wall-associated NlpC/P60 (or CHAP) cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that these two proteins act as gamma-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.

Structural Evidence That Scytalidolisin (Formerly Scytalidopepsin A) Is a Serine-Carboxyl Peptidase of the Sedolisin Family

Scytalidopepsin A, a pepstatin-insensitive acid endopeptidase from the fungus Scytalidium lignicolum was found to be a member of the sedolisin family of serine-carboxyl peptidases through analyses of the amino acid sequences of peptides derived from the reduced, pyridylethylated enzyme by enzymatic digestion. Hence it should be renamed scytalidolisin (or Scytalidium sedolisin).

Enhanced sequence coverage of proteins in human cerebrospinal fluid using multiple enzymatic digestion and linear ion trap LC-MS/MS

The cerebrospinal fluid (CSF) provides a ready access into the health state of the central nervous system, and alterations in some CSF proteins have been documented in brain disease. However, the complete variety of proteins is not known and methods to identify protein components are still being developed. The goal of this study was to examine the sequence coverage obtained from human CSF digests produced with different proteases. Enzymatic digests of CSF proteins were obtained with arginine-C endopeptidase (ArgC), glutamic acid endopeptidase (GluC), chymotrypsin, trypsin and their combinations, and then examined using reverse phase chromatography and a Finnigan LTQ linear ion trap mass spectrometer. Peptide sequences were identified with BioWorks 3.1 and sequence coverage calculated for the 38 most confidently identified proteins. Trypsin and GluC yielded greater coverage than chymotrypsin, while ArgC had the least sequence coverage. Protein sequence coverage was affected only slightly over four orders of magnitude dynamic range of abundance. Combining the peptides derived from different proteases further increased the coverage. Maximal sequence coverage was achieved by combining digest results from both GluC and trypsin. These results have implications for future studies to identify CSF proteins and their post-translational modifications.

CHARACTERIZATION OF EXCRETORY/SECRETORY ENDOPEPTIDASE AND METALLO-AMINOPEPTIDASES FROM TAENIA CRASSICEPS METACESTODES

Cysticercosis is caused by Taenia spp. metacestodes, which must survive in the host tissues to complete their life cycle. Their survival depends on their control of host immune responses. Because many parasites use proteases to modulate host responses, we examined culture media from Taenia crassiceps metacestodes for protease activity using peptide substrates. We identified prominent aminopeptidase activity at neutral pH, which was inhibited by chelating agents and partially inhibited by the aminopeptidase inhibitor, bestatin. Endopeptidase substrates were optimally cleaved at slightly acidic pH and endopeptidase activity was inhibited by cysteine protease inhibitors. Gel filtration FPLC and subsequent visualization by silver staining revealed a metallo-aminopeptidase of molecular weight 21 kDa and cysteine proteases of Mr 70 and 64 kDA. Recombinant IL-2 was digested when incubated with parasite culture supernatants, but not with control media. IL-2 degradation was completely inhibited by 1,10 phenanthroline and partially inhibited by bestatin, suggesting that a metallo-aminopeptidase was responsible. Incubation of human IgG with culture supernatants resulted in complete degradation of IgG, which was blocked by cysteine protease inhibitors. These observations demonstrate that Taenia spp. metacestodes secrete a number of proteolytic enzymes, which may target molecules from the host immune system and assist in evasion of the host immune response.

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide: IDENTIFICATION OF CRITICAL SEQUENCES AND RESIDUES IN THE PROPEPTIDE

Aspergilloglutamic peptidase (formerly called aspergillopepsin II) is an acid endopeptidase produced by Aspergillus niger var. macrosporus, with a novel catalytic dyad of a glutamic acid and a glutamine residue, thus belonging to a novel peptidase family G1. The mature enzyme is generated from its precursor by removal of the putative 41-residue propeptide and an 11-residue intervening peptide through autocatalytic activation. In the present study, the propeptide (Ala1-Asn41) and a series of its truncated peptides were chemically synthesized, and their effects on the enzyme activity and thermal stability were examined to identify the sequences and residues in the propeptide most critical to the inhibition and thermal stabilization. The synthetic propeptide was shown to be a potent competitive inhibitor of the enzyme (Ki = 27 nM at pH 4.0). Various shorter propeptide fragments derived from the central region of the propeptide had significant inhibitory effect, whereas their Ala scan-substituted peptides, especially R19A and H20A, showed only weak inhibition. Substitution of the Pro23-Pro24 sequence near His20 with an Ala-Ala sequence changed the peptide Lys18-Tyr25 to a substrate with His20 as the P1 residue. Furthermore, the propeptide was shown to be able to significantly protect the enzyme from thermal denaturation (DeltaTm = approximately 19 degrees C at pH 5.6). The protective potencies of the propeptide as well as truncated propeptides and their Ala scan-substituted peptides are parallel with their inhibitory potencies. These results indicate that the central part, and especially Arg19 and His20 therein, of the propeptide is most critical to the inhibition and thermal stabilization and that His20 interacts with the enzyme at or near the S1 site in a nonproductive fashion.

A proposed catalytic mechanism of aspergilloglutamic peptidase from Aspergillus niger

Aspergilloglutamic peptidase from Aspergillus niger (formerly called aspergillopepsin II) is a novel pepstatin-insensitive acid endopeptidase distinct from the well-studied aspartic peptidases, and thus is an interesting target for protein structure/function studies. Our recent X-ray crystallographic and site-directed mutagenesis studies showed that the enzyme is a glutamic peptidase in which glutamic acid-110 and glutamine-24 in the heavy chain are involved in the active site, forming a catalytic dyad. In the present study, we have investigated the pH-dependence of the enzyme activity and performed docking calculations to shed light on the catalytic mechanism of the enzyme. The results suggest a novel catalytic mechanism where glutamic acid-110 acts as a general acid in the first phase of catalysis. (Communicated by Masanori OTSUKA, M.J.A.)

Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Content and Degradation in Diploid, Tetraploid, and Hexaploid Wheat Species during Monocarpic Senescence

Wheat provides a unique genetic system in which variable sink size is available across the ploidies. We characterized monocarpic senescence in diploid, tetraploid, and hexaploid wheat species in flag leaf from anthesis up to full grain maturity at regular intervals. Triticum tauschii Acc. cv. EC-331751 showed the fastest rate of senescence among the species studied and the rate of loss per day was highest in terms of photosynthesis rate, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) content, and flag leaf N content coupled with a higher rate of gain in grain N content. Cultivars Kundan and HD 4530 maintained high flag leaf N content throughout grain filling as compared to the diploids and showed a slower rate of senescence. RuBPCO content was higher in the diploids as compared to Kundan and HD 4530 at anthesis. However, the rate of decline in RuBPCO content per day was also higher in the diploids. This degradation in RuBPCO was mediated by high endoproteolytic activities in the diploids which in turn supported its higher rate of N mobilization as compared to the tetraploid and hexaploid wheat. Acidic endopeptidases were responsible for the mobilization of flag leaf nitrogen in wheat across ploidy levels (r=−0.582, p<0.01).

The three-dimensional structure of aspergilloglutamic peptidase from Aspergillus niger

Aspergilloglutamic peptidase from Aspergillus niger is a novel pepstatin-insensitive acid endopeptidase distinct from the well-studied aspartic peptidases, and thus is an interesting target for protein structure/function studies. In the present study, we have determined the three-dimensional structure of the enzyme by X-ray crystallography to a 1.4-A resolution. The results revealed that the enzyme has a unique structure, composed of two seven-stranded anti-parallel β-sheets which form a β-sandwich structure and appear to have a partial two-fold symmetry, suggesting its possible evolution by gene duplication and that the glutamic acid-110 and glutamine-24 in the heavy chain form a catalytic dyad, consistent with our results obtained by site-directed mutagenesis. (Communicated by Masanori OTSUKA, M.J.A.)

Amidase domains from bacterial and phage autolysins define a family of γ- d, l-glutamate-specific amidohydrolases

Several phage-encoded peptidoglycan hydrolases have been found to share a conserved amidase domain with a variety of bacterial autolysins ( N-acetylmuramoyl- l-alanine amidases), bacterial and eukaryotic glutathionylspermidine amidases, γ- d-glutamyl- l-diamino acid endopeptidase and NLP/P60 family proteins. All these proteins contain conserved cysteine and histidine residues and hydrolyze γ-glutamyl-containing substrates. These cysteine residues have been shown to be essential for activity of several of these amidases and their thiol groups apparently function as the nucleophiles in the catalytic mechanisms of all enzymes containing this domain. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) superfamily includes a variety of previously uncharacterized proteins, including the tail assembly protein K of phage λ. Some members of this superfamily are important surface antigens in pathogenic bacteria and might represent drug and/or vaccine targets.

Endosomal Proteolysis of Internalized Insulin at the C-terminal Region of the B Chain by Cathepsin D

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.

Identification of insulin domains important for binding to and degradation by endosomal acidic insulinase.

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase (EAI), which hydrolyzes internalized insulin at a limited number of sites. Although the positions of these cleavages are partially known, the residues of insulin important in its binding to and proteolysis by EAI have not been defined. To this end, we have studied the degradation over time of native human insulin and three insulin-analog peptides using a soluble endosomal extract from rat liver parenchyma followed by purification of the products by HPLC and determination of their structure by mass spectrometry. We found variable rates of ligand processing, i.e. high ([Asp(B10)]- and [Glu(A13),Glu(B10)]-insulin), moderate (insulin) and low (the H2-analog). On the basis of IC(50) values, competition studies revealed that human and mutant insulins display nearly equivalent affinity for the EAI. Proteolysis of human and mutant insulins by EAI resulted in eight cleavages in the B-chain which occurred in the central region (Glu(B13)-Leu(B17)) and at the C-terminus (Arg(B22)-Thr(B27)), the latter region comprising the initial cleavages at Phe(B24)-Phe(B25) (major pathway) and Phe(B25)-Tyr(B26) (minor pathway) bonds. Except for the [Glu(A13),Glu(B10)]-insulin mutant, only one cleavage on the A-chain was observed at residues Gln(A15)-Leu(A16). Analysis of the nine cleavage sites showed a preference for hydrophobic and aromatic amino acid residues on both the carboxyl and amino sides of a cleaved peptide bond. Using the B-chain alone as a substrate resulted in a 30-fold increase in affinity for EAI and a 6-fold increase in the rate of hydrolysis compared with native insulin. A similar role for the C-terminal region of the B-chain of insulin in the high-affinity recognition of EAI was supported by the use of the corresponding B(22)-B(30) peptide, which displayed an increase in EAI affinity similar to the entire B-chain vs. wild-type insulin. Thus, we have identified a highly specific molecular interaction of insulin with EAI at the aromatic locus Phe(B24)-Phe(B25)-Tyr(B26). Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized [(125)I]Tyr(A14)-H2-analog, a protease-resistant insulin analog, undergoes a greater lysosomal transfer and lesser degradation than [(125)I]Tyr(A14)-insulin, confirming that endosomal sorting is regulated directly or indirectly by endosomal proteolysis.

Cathepsin D in normal and neoplastic thyroid tissues.

Cathepsin D is a widely distributed lysosomal acidic endopeptidase. It is an estrogen-regulated protein that is a prognostic factor in breast cancer. The aim of this study was to measure cathepsin D concentrations in thyroid tissues and to correlate these concentrations with clinical and pathologic parameters. Cathepsin D and thyroglobulin concentrations were measured in the cytosol of normal thyroid tissues (n = 14), benign nodules (n = 6), and thyroid carcinomas (n = 32) with an immunoradiometric assay. Statistical analysis was based on the Kruskal-Wallis and Wilcoxon tests and on the Spearman rank correlation coefficient. The mean level of cathepsin D, expressed as picomoles per milligram protein minus thyroglobulin, was higher in the 32 carcinomas, 29.1 +/- 15.5, than in the 14 normal thyroid tissues, 8.4 +/- 2.5 (p < 0.001) or in the 6 benign nodules, 11.2 +/- 7.3 (p = 0.003). Cathepsin D concentrations correlated with tumor size; Spearman rank correlation coefficient was rs = 0.44 (p = 0.012). No significant difference was found regarding histologic type. Cathepsin D concentrations were inversely correlated with the thyroglobulin level in the tumor; Spearman rank correlation coefficient was rs = -0.60 (p < 0.001). Cathepsin D concentration is higher in thyroid carcinoma than in normal thyroid tissue. Increased cathepsin D concentrations correlate with thyroid tumor size but not with histologic type. Further studies should be done to confirm the potential prognostic value of cathepsin D in patients with thyroid carcinomas.