Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 11, pp. 1667-1677 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 11, pp. 1781-1793.

1667

Serine Peptidase Homolog from

the Beetle Tenebrio molitor with Substitution

of Serine Residue with Threonine in the Catalytic Triad

Nikita I. Zhiganov

1,2

, Anna S. Gubaeva

, Valeriia F. Tereshchenkova

Yakov E. Dunaevsky

, Mikhail A. Belozersky

, and Elena N. Elpidina

1,a

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119992 Moscow, Russia

Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

e-mail: elp@belozersky.msu.ru

Received June 18, 2025

Revised October 8, 2025

Accepted October 15, 2025

Abstract— Analysis of the genomes and transcriptomes of the beetle Tenebrio molitor revealed a group of six

serine peptidase homologs (SPH) of the S1A chymotrypsin subfamily containing a conservative substitution of

the catalytic residue Ser195 with Thr (Ser195Thr) in the active center. All six SPH are secreted proteins with

prepropeptides and lack regulatory domains in the propeptide. The most highly expressed homolog, SerPH122,

shares 57% sequence identity with the most highly expressed elastase-like peptidase of T.  molitor, SerP41. Both

proteins exhibit similar domain organization, localization in the posterior midgut, and expression patterns

in the feeding stages of the fourth instar larva and imago. Testing hydrolytic activity of the recombinant

rSerPH122 preparation demonstrated that the conservative substitution of Ser for Thr in the active center

did not abolish its catalytic activity. rSerPH122 exhibits low specific activity but broad substrate specificity,

most effectively hydrolyzing substrates of chymotrypsin-like and trypsin-like peptidases. The homolog has a

pH optimum at 8.5 and is stable in the pH range 4.0-8.0. This study addresses the question of activity of the

homologs with the Ser195Thr substitution and contributes to understanding of the poorly studied area of

SPH functions, providing a basis for elucidating relationship between the structure and function of serine

peptidases and their homologs.

DOI: 10.1134/S0006297925601765

Keywords: serine peptidases, peptidase homologs, insect peptidases, Tenebrio molitor

* To whom correspondence should be addressed.

INTRODUCTION

Serine peptidases (SP) of the S1 family are the

most widespread and numerous group of peptidases,

both in terms of the number of sequenced pro-

teins and diversity of peptidase activities [1]. All ac-

tive peptidases in the S1 family are endopeptidases

and contain a catalytic triad consisting of His57,

Asp102, and Ser195 (residue numbering according

to bovine chymotrypsinogen  A, XP_003587247). Their

specificity largely depends on composition of the

S1  substrate-binding subsite at positions 189, 216,

and 226 [2]. The family also includes a significant

number of poorly studied proteins containing substi-

tutions of active center residues, commonly referred

to as inactive serine peptidase homologs (or pseudo-

peptidases), as they are presumed to lack catalytic ac-

tivity [3]. All animal peptidases of this family belong

to the S1A chymotrypsin subfamily [1, 4], which is

the most numerous and well-studied subfamily, in-

cluding enzymes such as trypsin, chymotrypsin, elas-

tase, and kallikrein. These enzymes play crucial roles

in digestion, blood coagulation, immune response,

and other physiological processes.

ZHIGANOV et al.1668

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Genomic and transcriptomic sequencing meth-

ods, which have become pivotal in recent years, are

particularly intensively used to characterize insects,

including within the international i5K project to

sequence the genomes of 5000 insects [5]. To date,

95 insect genomes from over 20 orders have been

sequenced (https://www.ncbi.nlm.nih.gov/datasets/

genome/?bioproject=PRJNA163993), and general char-

acterization of the sequenced genomes has shown that

most groups of insects contain a very large number

of proteins from the S1A chymotrypsin subfamily (up

to 300 or more). This makes insects an attractive ob-

ject for studying these proteins. However, large num-

ber of serine peptidases complicates analysis of these

protein groups, and such analysis has been conducted

only for the most thoroughly studied insect species,

most of which are harmful pests or model organisms:

dipterans Drosophila melanogaster [6] and Anopheles

gambiae [7]; hymenopterans Apis mellifera [8] and

Pteromalus puparum [9]; lepidopterans Bombyx mori

[10], Plutella xylostella [11], and Manduca sexta [12];

and beetles Tenebrio molitor [13] and Tribolium cas-

taneum [14]. It should be noted that in majority of

these studies, special attention was paid to the domain

organization of peptidases and the presumed role of

regulatory domains, while the presumed specificity of

peptidases was practically not analyzed.

Nevertheless, analysis of the available data has

shown that the maximum number of proteins from

the S1A chymotrypsin subfamily is found in the evo-

lutionarily young, intensively developing orders of

beetles and dipterans, among which a large number

of serine peptidase homologs (SPH) have been iden-

tified. For example, among the 337 proteins of the

S1A subfamily in the mosquito A.  gambiae, 117 were

SPH [7], while in the beetle T.  molitor, the number

of SPH was even higher – 125 SPH out of 269 pro-

teins of the S1A subfamily [13]. However, studies of

SPH are scarce. Most studies on the insect SPH are

devoted to bioinformatic analysis of their domain or-

ganization and analysis of gene expression at various

developmental stages and in different organs, which

has suggested their important role in insect metabo-

lism [7, 9]. There are also a small number of studies

characterizing individual insect SPH, most of which

contain the regulatory CLIP domain. Such homologs,

containing substitutions of the catalytic serine with

glycine and an S1 substrate-binding subsite character-

istic of trypsins, have been isolated from T.  molitor,

M.  sexta, and a number of other insects and crabs,

and their involvement in the cascade of prophenolox-

idase (PPO) activation reactions, which are associated

with immune response to bacterial or fungal infection,

has been shown [15-18]. It is assumed that their role

is to mediate protein interactions between the active

peptidases and their substrates or to participate in

localization of the members of peptidase cascades on

the surface of pathogens or parasites [19]. However,

the role of most SPHs, even in the well-studied model

insects, remains unknown. There is a detailed study

of spatial structure in combination with enzymatic

analysis of two representatives of the family from 32

SPH (SMIPPs) of the scabies mite Sarcoptes scabiei,

which indicates that SMIPPs have lost their ability to

bind substrates in the classical “canonical” manner

and instead have developed alternative functions in

the life cycle of the scabies mite [20]. It is believed

that SMIPPs have a protective function and inhibit

the host’s complement system, reducing its ability to

produce specific antibodies [21].

Among higher animals, there are only detailed

studies of human SPH azurocidin (or heparin-bind-

ing protein HBP, or cationic antimicrobial protein

37 kDa CAP37), which is close to neutrophil elastase,

has a broad spectrum of antimicrobial activity, binds

heparin, and is a multifunctional mediator of inflam-

mation [22]. The structure and role of SPH haptoglo-

bin, which binds free hemoglobin during hemolysis,

thereby removing it from circulation and preventing

oxidative tissue damage, have also been studied in

detail [23, 24]. In addition, the proteinZ (PZ), an SPH,

has been identified in the human blood plasma; it is

vitamin K-dependent and exists as a complex with

the serine peptidase inhibitor serpin, dependent on

protein Z (ZPI) [25]. The most important known phys-

iological function of PZ is its ability to enhance inhi-

bition of coagulation factor Xa by the inhibitor ZPI

by three orders of magnitude in the presence of a

phospholipid membrane and Ca

ions [25, 26].

It should be noted that lack of hydrolytic activity

in SPH has become generally accepted based on the

structural studies due to substitutions in the triad of

amino acid residues of the active center [3]. There

are very few biochemical studies dedicated to exam-

ining catalytic activity of SPH. Among the homologs

ofserine peptidases of the chymotrypsin S1A subfam-

ily, the studies showing absence of catalytic activity

were conducted with the isolated homolog azurocidin

[27] and SMIPPs of the scabies mite [20]. Similar re-

sults were obtained also for the TIN-ag-RP, a homolog

of the cysteine peptidase cathepsin B of the papain

C1 family [28].

Many SPH genes exhibit high levels of expression,

indicating their active involvement in the organism’s

metabolism. Researchers agree that the presence of

inactive enzyme homologs is expected and that an

inactive homolog usually evolved from an active en-

zyme precursor, not vice versa [3]. Inactive homologs

apparently evolved in parallel with their active en-

zymes and are currently considered important com-

ponents of biological systems. Such large number of

both inactive and active serine peptidases and their

SERINE PEPTIDASE HOMOLOG FROM Tenebrio molitor 1669

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

conservation in insects indicate their gene expansion,

and evolution of SPHs and their new functions sug-

gests that they are beneficial for insects [29]. Growing

importance and relevance of studying SPH are em-

phasized by their high abundance and wide range of

the processes in which they are involved. Of particu-

lar interest are SPHs from the organisms where the

ratio of active peptidases to inactive homologs is close

to one, as observed in the genomes of rapidly evolv-

ing insects. As more SPHs are identified, there is an

increasing need for deep understanding of their struc-

ture, phylogenetic relationships, and functional roles.

It should also be noted that the important regu-

latory function of enzyme homologs in metabolic and

signaling pathways, often associated with pathologi-

cal conditions, makes them potential targets for the

development of new therapeutic agents, which could

become a promising direction for future research

and allow identification of new regulatory chains,

expanding the possibilities for the search for new

drugs. Analysis of the available data will provide a

new perspective on the meaning of existence and con-

servation of the enzyme homologs during evolution

and outline new research directions aimed at under-

standing their functional role.

This article uses genomic and transcriptomic

approaches to characterize a group of SPHs from a

stored product pest, the yellow mealworm Tenebrio

molitor, with a conservative substitution of the cat-

alytic triad residue Ser for Thr and tests and char-

acterizes enzymatic activity of one of the homologs,

SerPH122.

MATERIALS AND METHODS

Analysis of amino acid sequences of T.  molitor

SPH with the conservative substitution Ser195Thr.

Multiple alignment of amino acid sequences of SPH

with the conservative substitution Ser195Thr was per-

formed using the MAFFT version 7 (https://mafft.cbrc.jp/

alignment/server/) [30]. Composition of the active cen-

ter and the S1 substrate-binding subsite was deter-

mined based on the bovine alpha-chymotrypsin [31].

Calculation of mRNA expression levels of SPHs

at different stages of the T. molitor life cycle. Ex-

pression profiles of mRNA of six SPHs (SerPH122,

SerPH79, SerPH245, SerPH342, SerPH395, SerPH486)

and four active gut serine peptidases (SerP1, SerP38,

SerP41, SerP69) were calculated with the formula

log

(RPKM  +  1) (where RPKM is Reads Per Kilobase

per Million mapped reads, +1 is used to perform cal-

culations at zero expression) using transcriptomes at

different stages of the T.  molitor life cycle (egg, II and

IV instar larvae, early and late pupae, imago). Prepa-

ration of biological material, RNA isolation, cDNA se-

quencing, assembly, and analysis of transcriptomes

were described earlier [13].

Obtaining of a recombinant proenzyme prepa-

ration of the homolog rSerPH122. Recombinant

preparation of the deglycosylated proenzyme of the

homolog rSerPH122 was obtained and purified us-

ing the previously published methods [32]. Recombi-

nant expression was carried out in the yeast system

Komagataella kurtzmanii. A 6-histidine tag (His

-tag)

was added to the C-terminus of the recombinant pro-

tein for further purification, which was performed us-

ing a specific metal-chelate affinity chromatography,

ensuring high selectivity and purity of the prepara-

tion.

Processing of the recombinant proenzyme

rSerPH122. The lyophilized preparation of the proen-

zyme of the homolog rSerPH122 was dissolved in

0.1  M acetate-phosphate-borate universal buffer (UB)

(pH  7.9) [33] at concentration of 15  µg/ml. Trypsin

was next added to a final concentration of 0.25  µg/ml,

after which the reaction mixture was incubated for

60 min at 37°C. After activation of the proenzyme,

an irreversible trypsin inhibitor, N-α-tosyl-L-lysine

chloromethyl ketone hydrochloride (TLCK), was add-

ed to the reaction mixture to a final concentration

of 3.3  nM to inhibit trypsin. Concentration of TLCK

required for complete inhibition of trypsin was deter-

mined by titration in a separate experiment.

Assay of enzymatic activity of the recombi-

nant preparation of rSerPH122 using chromogen-

ic substrates. Enzymatic activity was determined

based on the initial rate of hydrolysis of chromo-

genic peptide substrates containing a p-nitroaniline

residue (p-nitroanilide, pNA) for detection. In a mi-

croplate well, 5 to 20  µl of the recombinant prepa-

ration of rSerPH122 (concentration in the reaction

mixture – 0.9  µM) was added to each well, followed

by addition of a 20  mM Tris-HCl (pH  8.0) or 0.1  M  UB

(pH  7.9) to a final volume of 195  µl. Next, 5  µl of a

substrate solution in dimethylformamide (DMF) was

added. Standard substrate concentration in the reac-

tion mixture was 0.25  mM, and DMF concentration in

the reaction mixture was 2.5% vol. During the reac-

tion, the mixture was incubated at 37°C. The amount

of p-nitroaniline formed was determined in 96-well

plates (Medpolymer, Russia) with a microplate pho-

tometer ELx808 (BioTek Instruments, Inc., USA) by

measuring absorbance of the solution at 405 nm at

the initial time point and then every 5 min. The fol-

lowing substrates were used: trypsin-like peptidase

substrates – Z-FR-pNA, Z-RR-pNA, Bz-R-pNA; chymo-

trypsin-like – Suc-AAPF-pNA, Glp-AAF-pNA, Glp-F-pNA,

Suc-F-pNA, Ac-Y-pNA; elastase-like – MeOSuc-AAPV-

pNA, Suc-AAA-pNA; substrates hydrolyzed by both

chymotrypsin-like and elastase-like peptidases – Suc-

AAPL-pNA, Glp-AAL-pNA, For-AAL-pNA, Glp-FL-pNA;

ZHIGANOV et al.1670

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

cysteine peptidase substrates – Glp-FA-pNA, Glp-FQ-

pNA (where Z – benzyloxycarbonyl (protective group

of substrates); pNA – p-nitroanilide; Bz – benzoyl; Suc –

succinyl; Glp – pyroglutamyl; Ac – acetyl; For– formyl).

The following commercial substrates were used

in the study: Z-FR-pNA, Z-RR-pNA, Bz-R-pNA, Suc-

AAPF-pNA, MeOSuc-AAPV-pNA, Suc-AAA-pNA; Suc-

AAPL-pNA, Glp-AAL-pNA (Bachem, Switzerland);

Ac-Y-pNA (Serva, Germany). Substrates Glp-AAF-pNA,

Glp-F-pNA, Suc-F-pNA, For-AAL-pNA, Glp-FL-pNA,

Glp-FA-pNA were synthesized using standard methods

[34] at the Laboratory of Protein Chemistry, Depart-

ment of Chemistry of Natural Compounds, Faculty

of Chemistry, Lomonosov Moscow State University;

Glp-FQ-pNA was synthesized according to the method

of Filippova et al. [35].

Enzymatic activity was calculated using the for-

mula (1):

a = kdA

405

 / dt, (1)

where a is activity of the preparation, nmol/min;

k  =  31.9  nmol/optical unit – the amount of p-nitroan-

iline at which absorbance of the solution was equal

to 1 optical unit (the coefficient was determined in

a special experiment by constructing a calibration

curve of the dependence of absorbance of solutions

on concentration of p-nitroaniline), dA

405

/dt is the

change in absorbance of the solution at 405 nm at

time t, optical units/min.

Calculation of activity was performed using

Microsoft Office Excel based on the initial rate of

p-nitroaniline formation on the linear part of the ki-

netic curve with subtracting A

405

values at the initial

time point. Initial rates of hydrolysis were determined

as the tangents of the slopes of the linear parts of the

obtained dependences of optical absorbance on time.

Effect of pH on rSerPH122 activity and stabil-

ity. Effect of pH on rSerPH122 activity was assessed

during hydrolysis of the substrates Suc-AAPF-pNA and

Glp-AAF-pNA in 0.1M UB in the pH range from 3.0 to

11.0 with a step of 0.5, as described above.

To study pH stability, the rSerPH122 preparation

was incubated in 50  µl of 0.01  M  UB with pH from

2.5 to 11.0 and a step of 0.5 for 30  min. The pH in all

samples was then adjusted to 7.9 by adding 0.1  M  UB

(pH  7.9) to a final volume of 195  µl, and activity was

measured as described above.

Statistical data processing. Each experiment

with determination of enzymatic activity of the re-

combinant preparation of rSerPH122 and effect of pH

on its activity and stability was performed in at least

three replicates. Microsoft Excel 2013 was used for

statistical processing of the obtained data, employing

built-in functions to assess standard deviation and

confidence intervals using Student’s t-distribution

(significance level (alpha) – 0.1 was used to calculate

the confidence intervals).

RESULTS

Analysis of amino acid sequences of SPH with

the Ser195Thr substitution. A detailed analysis of the

genomes and transcriptomes of the beetle Tenebrio

molitor allowed to identify and annotate 269 amino

acid sequences of serine peptidases from the S1A chy-

motrypsin subfamily. Among these, 125 sequences had

1-3 substitutions of amino acid residues in the catalytic

triad (His57, Asp102, and Ser195) and were homologs

of serine peptidases (SPH) [13]. Analysis of the SPH

amino acid sequences revealed a group of six SPHs

containing His, Asp, and Thr residues in the active

site, with a conservative substitution of the catalytical-

ly essential Ser residue with Thr (Ser195Thr, HDT-type

substitution) (Fig. 1; Table 1). All six SPHs contained

pre- and propeptides, indicating that they are secreted

proteins and, based on bioinformatics analysis, lack

regulatory domains in the propeptide [13].

mRNA expression of the SPHs with Ser195Thr

substitution. Analysis of mRNA expression across six

life stages of the beetle – eggs, II and IV instar larvae,

early and late pupae, and adults – revealed a similar

expression profile for all six SPHs, with the highest

levels observed for the SerPH122 (Fig. 2). mRNA ex-

pression was detected almost exclusively during the

feeding stages (IV instar larvae and adults). A similar

expression profile was observed for the elastase-like

SerP41. In contrast, the mRNAs for trypsin SerP1

and chymotrypsin-like peptidase SerP69 were also

expressed in the II instar larvae, while the digestive

chymotrypsin-like peptidase SerP38 showed low ex-

pression in adults.

Substrate specificity of rSerPH122. The recom-

binant proenzyme, rSerPH122, was produced in the

yeast strain K.  kurtzmanii, purified using metal-che-

late affinity chromatography, and deglycosylated as

previously described [32]. Given the conservative

Ser195Thr substitution in the catalytic triad, we inves-

tigated catalytic activity of the recombinant rSerPH122

processed with trypsin using the chymotrypsin sub-

strate Suc-AAPF-pNA [32]. Notably, the initial recom-

binant rSerPH122 preparation, produced with the

pro-sequence, exhibited activity after a few minutes

of incubation, suggesting potential autoprocessing.

This is in agreement with the subsequent findings

on substrate specificity – enzymatic activity with the

substrates containing an arginine residue at the P1

position (Fig. 3). Additional processing with trypsin

increased protein activity by only 1.5-fold. To avoid po-

tential cross-influence of trypsin on the experimental

results (despite the use of titrated TLCK inhibitor

SERINE PEPTIDASE HOMOLOG FROM Tenebrio molitor 1671

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Fig.  1. Multiple sequence alignment of T. molitor sequences for four active digestive serine peptidases (trypsin SerP1,

chymotrypsin-like SerP38 and SerP69, and elastase-like SerP41) and six SPHs (SerPH79, SerPH122, SerPH245, SerPH342,

SerPH395, SerPH486) with Ser195Thr substitution in the active site. The catalytic triad residues are highlighted in green,

the S1 substrate-binding subsite residues in yellow, signal peptides in purple, the C-terminal residue of the propeptide at

the processing site in blue, and first four N-terminal residues of the peptidase domain in gray.

ZHIGANOV et al.1672

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Table 1. Composition of the active site and S1 substrate-binding subsite of HDT-type SPHs and major digestive

peptidases of T. molitor

Sequences no. NCBI ID Processing site Active center

S1 substrate-

binding subsite

% of identity

with SerPH122

SerPH122 CAH1368241 R|IIGG H D T G L S 100

SerPH79 CAH1375498 R|IIGG H D T G M T 47

SerPH245 CAH1375497 R|IIGG H D T G M T 48

SerPH342 KAJ3632571 R|IIGG H D T G I K 51

SerPH395 KAJ3632570 R|IIGG H D T G I K 53

SerPH486 CAH1375496 R|IIGG H D T G F S 48

SerP41 ABC88760 R|IVGG H D S G I S 57

SerP69 ABC88746 R|IISG H D S S G S 37

SerP38 QRE01764 R|VVGG H D S G G D 30

SerP1 ABC88729 R|IVGG H D S D G G 31

Fig.  2. mRNA expression profiles of six SPHs (SerPH79, SerPH122, SerPH245, SerPH342, SerPH395, SerPH486) and four active

digestive serine peptidases (trypsin SerP1, chymotrypsin-like SerP38 and SerP69, and elastase-like SerP41) at different life

stages of T. molitor. Expression levels are presented as log

(RPKM + 1) values for each stage, with error bars indicating

standard deviation (SD).

at the end of processing), the unprocessed homolog

preparation was used to study substrate specificity.

Similar results were obtained also with the processed

enzyme preparation.

In this study, we analyzed substrate specificity of

the rSerPH122 using a broad range of chromogenic

peptide substrates for trypsin-like, chymotrypsin-like,

elastase-like, and cysteine peptidases (Fig. 3). Prefer-

ential cleavage of the chymotrypsin-like substrates

(Suc-AAPF-pNA, Glp-AAF-pNA, Glp-F-pNA, Ac-Y-pNA)

was observed, along with significant activity with

the trypsin-like substrates (Z-FR-pNA and Z-RR-pNA).

It should be noted that hydrolysis of the Z-FR-pNA

substrate stopped after 20min, despite sufficient sub-

strate remaining in the reaction mixture, unlike the

hydrolysis of Glp-AAF-pNA (Fig.4). Addition of trypsin

to this reaction mixture resulted in further cleavage

of the original substrate.

SERINE PEPTIDASE HOMOLOG FROM Tenebrio molitor 1673

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Fig.  3. Activity of the recombinant rSerPH122 homolog with various substrates. Assay conditions: 0.9 µM rSerPH122,

0.25mM chromogenic substrate, 20mM Tris-HCl (pH 8.0), and 2.5% (v/v) DMF. Error bars represent standard deviation (SD).

Fig.  4. Kinetic curves of the rSerPH122-mediated hydrolysis of Z-FR-pNA (blue) and Glp-AAF-pNA (purple) substrates. Error

bars represent standard deviation (SD).

Comparison of the specific activities of recombi-

nant preparations – rSerPH122 and the chymotryp-

sin-like peptidase rSerP38 [36] (expressed at compa-

rable levels in the IV instar T. molitor larvae) was

conducted using the chymotrypsin substrate Suc-AAPF-

pNA. Activity of the homolog was significantly lower:

11nmol/min/mg compared to 49,000  nmol/min/mg for

the active SerP38 peptidase.

Effect of pH on the rSerPH122 activity and

stability. pH dependence of activity and stability of

the rSerPH122 was investigated (Fig. 5). The homo-

log exhibited maximal activity in the alkaline pH

range with optimum at pH 8.5 (Fig. 5a), similar to

the rSerP38. Moreover, like rSerP38, the homologue

demonstrated the highest stability (retaining 85-100%

of activity) in the pH range 4.0-8.0 (Fig. 5b).

DISCUSSION

Among the 125 SPH sequences homologous to

serine peptidases of the S1A chymotrypsin subfamily

in the T.  molitor genome [13], six SPHs were found

to contain a conservative Ser195 to Thr substitution

ZHIGANOV et al.1674

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

Fig. 5. Effect of pH on activity (a) and stability (b) of the recombinant rSerPH122 homolog (0.9 µM). Error bars represent

the confidence interval.

in the catalytic triad. All homologs with the HDT-type

substitution are represented by sequences containing

pre- and propeptides, are secreted, and lack regula-

tory domains in the propeptide. These homologs are

processed to their mature form by trypsin at the ar-

ginine residue at the C-terminus of the propeptide, as

demonstrated for the most highly expressed protein

in this group, SerPH122 [32]. Additionally, our data

on the substrate specificity of SerPH122 (its ability

to hydrolyze substrates containing arginine at the P1

position) and its proteolytic activity prior to process-

ing with trypsin suggest its capacity for autoprocess-

ing, a distinctive feature of this homolog. Sequence

identity comparisons revealed that SerPH122 shares

the highest similarity not with other homologs in this

group but with the most highly expressed elastase-like

peptidase, SerP41 (57%), indicating a potential evolu-

tionary relationship. This is further supported by the

similar expression profiles of the HDT-type homologs

and SerP41 – only during the feeding stages of the IV

instar larvae and adults, whereas the main digestive

peptidases, trypsin SerP1 and chymotrypsin-like pep-

tidase SerP69, are also expressed during the feeding

II instar larvae, and the digestive chymotrypsin-like

peptidase SerP38 exhibits low expression in adults.

It is worth noting that the SPH azurocidin is also

closely related to the neutrophil elastase [22].

Considering the conservative Ser195Thr substitu-

tion in the HDT-type homolog group, we proceeded

to investigate enzymatic activity of the most highly

expressed homolog, SerPH122, which was also the

only one from this group reliably detected in the

gut extract of the fourth-instar T. molitor larvae [37].

Unlike the vast majority of the studied inactive pep-

tidase homologs with substitutions in key active site

residues [3], the rSerPH122 retained weak but detect-

able proteolytic activity against a broad range of ser-

ine and cysteine peptidase substrates with preference

for trypsin and chymotrypsin substrates. Interestingly,

hydrolysis of the trypsin substrate Z-FR-pNA began at

the highest rate among the all-tested substrates but

ceased after 20 min, despite the remaining substrate

in the reaction mixture. One possible cause for the

reaction stopping could be negative effect of the re-

action products on the SerPH122 activity. We note

that the detected hydrolytic activities of rSerPH122

were low, and its activity against the chymotrypsin

substrate was 4500 times lower than that of the di-

gestive chymotrypsin-like peptidase rSerP38 [36]. This

suggests that the conservative Ser195Thr substitution

impairs full functionality of SerPH122 as an active en-

zyme for hydrolyzing dietary proteins and implies a

different role for this protein in the digestive process.

The alkaline pH optimum (pH8.5) and maximum sta-

bility at pH 4.0-8.0 for SerPH122, similar to rSerP38,

correlate with the fact that SerPH122 and SerP38 are

most abundant in the posterior midgut (PM) of T. mo-

litor [37], where the average pH is 7.9 [38].

Homologs with the Ser195Thr substitution are

found almost exclusively in insects, and their char-

acterization is of particular interest, as they have

been proposed as a likely transitional form during

the evolutionary switch between two types of serine

codons: TCX and AGX, which theoretically can only

occur through an intermediate protein containing

a Ser195 to Thr or Cys substitution due to a single

base change (ACX) [1]. Only Thr has been found in

this position in the SPH sequences. The authors of

this hypothesis, Rawlings and Barrett [1], questioned

whether such intermediate form would retain ac-

tivity. Our positive answer to this question makes

the authors’ assumption about the existence of such

SERINE PEPTIDASE HOMOLOG FROM Tenebrio molitor 1675

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

intermediate form highly probable in connection with

the expansion of the functional capabilities of these

intermediate protein forms.

Combining data from our laboratory on localiza-

tion [37] and high expression of the gene encoding

SerPH122 in the gut of fourth-instar T.  molitor larvae,

as well as its significant activity with the substrates

that have arginine at the P1 position and its abili-

ty to autoprocessing at this residue, we cannot ex-

clude involvement of this homolog in activation of

the digestive enzymes through processing, as most

serine peptidases in T. molitor are processed at argi-

nine residues [13]. Broad substrate specificity could

also provide greater flexibility in the interaction of

rSerPH122 with various protein partners, facilitating

its participation in diverse metabolic processes. It is

noteworthy that knockdown of the gene encoding the

main digestive cysteine peptidase in the closely relat-

ed beetle Tribolium castaneum leads to the increased

expression of some SPHs alongside with the active

digestive SPs [39], indicating their important protec-

tive role.

This work expands our knowledge on the func-

tional roles of SPHs and could serve as a basis for

elucidating relationship between the structure and

function of serine peptidases and their homologs.

CONCLUSION

Emergence of the homologs of various enzymes

is likely an evolutionary advantage, adding new reg-

ulatory levels to complex networks. Our transcrip-

tomic studies have shown that SPH genes are active-

ly transcribed, particularly during the feeding stages

of larvae and adults. Specifically, SerPH122, localized

in the gut of T.  molitor larvae, exhibits high expres-

sion during feeding stages, as well as autoprocessing

ability and broad substrate specificity. This suggests

its involvement in the digestive process, possibly

through regulation or activation of other digestive

peptidases. The obtained data highlight the important

role of SerPH122 in the digestive system of T. moli-

tor and emphasize the need for further research

to understand its precise function and regulatory

mechanisms.

Abbreviations

Ac acetyl

Bz benzoyl

DMF dimethylformamide

For formyl

Glp pyroglutamyl

pNA p-nitroanilide

SerP SP serine peptidase

SerPH SPH serine peptidase homolog

Suc succinyl

TLCK

N-α-Tosyl-L-lysine chloromethyl ketone

hydrochloride

UB universal buffer

benzyloxycarbonyl (protecting group

for substrates)

Contributions

E. N. Elpidina and V. F. Tereshchenkova – concept

and supervision; N. I. Zhiganov and A. S. Gubaeva –

experiments; Ya. E. Dunayevsky and M. A. Belozer-

sky – discussion of results; Ya. E. Dunayevsky and

E. N. Elpidina – manuscript writing; V. F. Tereshchen-

kova– manuscript editing.

Funding

This study was conducted under the State Assign-

ment of Lomonosov Moscow State University (projects

nos.121031300037-7 and 123063000002-7).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

REFERENCES

1. Rawlings, N. D., and Barrett, A. J. (2013) Introduction:

Serine Peptidases and Their Clans. Handbook of Pro-

teolytic Enzymes, (Rawlings, N. D., and Salvesen, G.,

eds) 3rd Edn, Elsevier Ltd., pp. 2491-2523, https://

doi.org/10.1016/B978-0-12-382219-2.00559-7.

2. Schechter, I., and Berger, A. (1967) On the size of the

active site in proteases. I. Papain, Biochem. Biophys.

Res. Commun., 27, 157-162, https://doi.org/10.1016/

S0006-291X(67)80055-X.

3. Reynolds, S.L., and Fischer,K. (2015) Pseudoproteas-

es: mechanisms and function, Biochem.J., 468, 17-24,

https://doi.org/10.1042/BJ20141506.

4. Rawlings, N. D., and Bateman, A. (2021) How to use

the MEROPS database and website to help under-

stand peptidase specificity, Protein Sci., 30, 83-92,

https://doi.org/10.1002/pro.3948.

5. Levine, R. (2011) i5k: the 5,000 insect genome proj-

ect, Am.Entomol., 57, 110-113, https://doi.org/10.1093/

ae/57.2.110.

6. Ross, J., Jiang, H., Kanost, M., and Wanga, Y. (2003)

Serine proteases and their homologs in the Drosoph-

ila melanogaster genome: an initial analysis of se-

quence conservation and phylogenetic relationships,

Gene, 304, 117-131, https://doi.org/10.1016/s0378-

1119(02)01187-3.

ZHIGANOV et al.1676

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

7. Cao, X., Gulati, M., and Jiang, H. (2017) Serine prote-

ase-related proteins in the malaria mosquito, Anoph-

eles gambiae, Insect Biochem. Mol. Biol., 88, 48-62,

https://doi.org/10.1016/j.ibmb.2017.07.008.

8. Zou, Z., Lopez, D. L., Kanost, M. R., Evans, J. D., and

Jiang, H. (2006) Comparative analysis of serine pro-

tease-related genes in the honey bee genome: possi-

ble involvement in embryonic development and in-

nate immunity, Insect Mol. Biol., 15, 603-614, https://

doi.org/10.1111/j.1365-2583.2006.00684.x.

9. Yang, L., Lin, Z., Fang, Q., Wang, J., Yan, Z., Zou, Z.,

Song,Q., and Ye,G. (2017) Thegenomic and transcrip-

tomic analyses of serine proteases and their homo-

logs in an endoparasitoid, Pteromalus puparum, Dev.

Comp. Immunol., 77, 56-68, https://doi.org/10.1016/

j.dci.2017.07.014.

10. Zhao, P., Wang, G. H., Dong, Z. M., Duan, J., Xu,

P. Z., Cheng, T. C., Xiang, Z. H., and Xia, Q. Y. (2010)

Genome-wide identification and expression analy-

sis of serine proteases and homologs in the silk-

worm Bombyx mori, BMC Genomics, 11, 405, https://

doi.org/10.1186/1471-2164-11-405.

11. Lin,H., Xia,X., Yu,L., Vasseur,L., Gurr, G.M., Yao,F.,

Yang, G., and You, M. (2015) Genome-wide identifi-

cation and expression profiling of serine proteases

and homologs in the diamondback moth, Plutel-

la xylostella (L.), BMC Genomics., 16, 1054, https://

doi.org/10.1186/s12864-015-2243-4.

12. Cao, X., He, Y., Hu, Y., Zhang, X., Wang, Y., Zou, Z.,

Chen, Y., Blissard, G. W., Kanost, M. R., and Jiang, H.

(2015) Sequence conservation, phylogenetic relation-

ships, and expression profiles of nondigestive serine

proteases and serine protease homologs in Manduca

sexta, Insect Biochem. Mol. Biol., 62, 51-63, https://

doi.org/10.1016/j.ibmb.2014.10.006.

13. Zhiganov, N. I., Vinokurov, K. S., Salimgareev, R. S.,

Tereshchenkova, V. F., Dunaevsky, Y. E., Belozersky,

M. A., and Elpidina, E. N. (2024) The set of serine

peptidases of the Tenebrio molitor beetle: transcrip-

tomic analysis on different developmental stages,

Int.J. Mol. Sci., 25, 5743, https://doi.org/10.3390/

ijms25115743.

14. Cao, X., and Jiang, H. (2018) Building a platform

for predicting functions of serine protease-related

proteins in Drosophila melanogaster and other in-

sects, Insect Biochem. Mol. Biol., 103, 53-69, https://

doi.org/10.1016/j.ibmb.2018.10.006.

15. Kwon, T. H., Kim, M. S., Choi, H. W., Joo, C. H., Cho,

M. Y., and Lee, B. L. (2000) A masquerade-like ser-

ine proteinase homologue is necessary for phenolox-

idase activity in the coleopteran insect, Holotrichia

diomphalia larvae, Eur. J. Biochem., 267, 6188-6196,

https://doi.org/10.1046/j.1432-1327.2000.01695.x.

16. Lee, K. Y., Zhang, R., Kim, M. S., Park, J. W., Park,

H. Y., Kawabata, S., and Lee, B. L. (2002) A zymo-

gen form of masquerade-like serine proteinase ho-

mologue is cleaved during pro-phenoloxidase ac-

tivation by Ca

in coleopteran and Tenebrio moli-

tor larvae, Eur. J. Biochem., 269, 4375-4383, https://

doi.org/10.1046/j.1432-1033.2002.03155.x.

17. Gupta, S., Wang, Y., and Jiang, H. (2005) Manduca

sexta prophenoloxidase (proPO) activation requires

proPO-activating proteinase (PAP) and serine pro-

teinase homologs (SPHs) simultaneously, Insect Bio-

chem. Mol. Biol., 35, 241-248, https://doi.org/10.1016/

j.ibmb.2004.12.003.

18. Liu, H. P., Chen, R. Y., Zhang, M., and Wang, K. J.

(2010) Isolation, gene cloning and expression profile

of a pathogen recognition protein: a serine protein-

ase homolog (Sp-SPH) involved in the antibacterial

response in the crab Scylla paramamosain, Dev.

Comp. Immunol., 34, 741-748, https://doi.org/10.1016/

j.dci.2010.02.005.

19. Kanost, M. R., and Jiang, H. (2015) Clip-domain

serine proteases as immune factors in insect he-

molymph, Curr. Opin. Insect Sci., 11, 47-55, https://

doi.org/10.1016/j.cois.2015.09.003.

20. Fischer,K., Langendorf, C.G., Irving, J.A., Reynolds,S.,

Willis,C., Beckham,S., Law, R.H., Yang,S., Bashtannyk-

Puhalovich, T. A., McGowan, S., Whisstock, J. C., Pike,

R. N., Kemp, D. J., and Buckle, A. M. (2009) Structur-

al mechanisms of inactivation in scabies mite ser-

ine protease paralogues, J. Mol. Biol., 390, 635-645,

https://doi.org/10.1016/j.jmb.2009.04.082.

21. Reynolds, S. L., Pike, R. N., Mika, A., Blom, A. M.,

Hofmann, A., Wijeyewickrema, L. C., Kemp, D., and

Fischer, K. (2014) Scabies mite inactive serine pro-

teases are potent inhibitors of the human comple-

ment lectin pathway, PLoS Negl. Trop. Dis., 8, e2872,

https://doi.org/10.1371/journal.pntd.0002872.

22. Watorek, W. (2003) Azurocidin – inactive serine

proteinase homolog acting as a multifunction-

al inflammatory mediator, Acta Biochim. Pol., 50,

743-752.

23. Kurosky, A., Barnett, D. R., Lee, T. H., Touchstone, B.,

Hay, R. E., Arnott, M. S., Bowman, B. H., and Fitch,

W. M. (1980) Covalent structure of human haptoglo-

bin: a serine protease homolog, Proc. Natl. Acad.

Sci. USA, 77, 3388-3392, https://doi.org/10.1073/

pnas.77.6.3388.

24. Andersen, C. B. F., Stødkilde, K., Sæderup, K. L.,

Kuhlee, A., Raunser, S., and Graversen, J. H. (2017)

Haptoglobin, Antioxid. Redox Signal., 26, 814-831,

https://doi.org/10.1089/ars.2016.6793.

25. Rezaie, A. R., Bae, J. S., Manithody, C., Qureshi,

S. H., and Yang, L. (2008) Protein Z-dependent pro-

tease inhibitor binds to the C-terminal domain of

protein Z, J.Biol. Chem., 283, 19922-19926, https://

doi.org/10.1074/jbc.M802639200.

26. Chandrasekaran, V., Lee, C. J., Duke, R. E., Perera, L.,

and Pedersen, L. G. (2008) Computational study of

the putative active form of proteinZ (PZa): sequence

SERINE PEPTIDASE HOMOLOG FROM Tenebrio molitor 1677

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

design and structural modeling, Protein Sci., 17,

1354-1361, https://doi.org/10.1110/ps.034801.108.

27. Campanelli, D., Detmers, P. A., Nathan, C. F., and

Gabay, J. E. (1990) Azurocidin and a homologous

serine protease from neutrophils. Differential anti-

microbial and proteolytic properties, J.Clin. Invest.,

85, 904-915, https://doi.org/10.1172/JCI114518.

28. Wex,T., Lipyansky,A., Brömme, N.C., Wex,H., Guan,

X. Q., and Brömme, D. (2001) TIN-ag-RP, a novel cat-

alytically inactive cathepsin B-related protein with

EGF domains, is predominantly expressed in vascu-

lar smooth muscle cells, Biochemistry, 40, 1350-1357,

https://doi.org/10.1021/bi002266o.

29. Pils, B., and Schultz, J. (2004) Inactive enzyme-ho-

mologues find new function in regulatory process-

es, J. Mol. Biol., 340, 399-404, https://doi.org/10.1016/

j.jmb.2004.04.063.

30. Katoh, K., Rozewicki, J., and Yamada, K. D. (2019)

MAFFT online service: multiple sequence alignment,

interactive sequence choice and visualization, Brief

Bioinform., 20, 1160-1166, https://doi.org/10.1093/

bib/bbx108.

31. Perona, J. J., and Craik, C. S. (1995) Structural ba-

sis of substrate specificity in the serine proteas-

es, Protein Sci., 4, 337-360, https://doi.org/10.1002/

pro.5560040301.

32. Tereshchenkova, V. F., Zhiganov, N. I., Akentiev, F. I.,

Gubaidullin, I. I., Kozlov, D. G., Belyaeva, N. V., Fi-

lipova, I. Y., and Elpidina, E. N. (2021) Preparation

and properties of the recombinant Tenebrio molitor

SerPH122 – proteolytically active homolog of serine

peptidase, Appl. Biochem. Microbiol., 57, 579-585,

https://doi.org/10.1134/S0003683821050161.

33. Frugoni, J.A.C. (1957) Tampone universale di Britton

e Robinson a forza ionica constante, Gazz. Chem.

Ital., 87, 403-407.

34. Goodman, M., Toniolo, C., Moroder, L., and Felix, A.

(2004) Houben-Weyl Methods of Organic Chemis-

try: Synthesis of Peptides and Peptidomimetics,

4th Edn., Vol. E22a, Stuttgart, NY, Thieme, https://

doi.org/10.1055/b-0035-112838.

35. Filippova, I. Y., Dvoryakova, E. A., Sokolenko, N. I.,

Simonyan, T. R., Tereshchenkova, V. F., Zhiganov,

N. I., Dunaevsky, Y. E., Belozersky, M. A., Oppert, B.,

and Elpidina, E. N. (2020) New glutamine-containing

substrates for the assay of cysteine peptidases from

the C1 papain family, Front. Mol. Biosci., 7, 578758,

https://doi.org/10.3389/fmolb.2020.578758.

36. Tereshchenkova, V. F., Zhiganov, N. I., Gubaeva,

A. S., Akentiev, F. I., Dunayevsky, Y. E., Kozlov, D. G.,

Belozersky, M. A., and Elpidina, E. N. (2024) Re-

combinant chymotrypsin-like peptidase from Tene-

brio molitor with a non-canonical substrate-binding

site, Appl. Biochem. Microbiol., 60, 420-430, https://

doi.org/10.1134/S0003683824603652.

37. Zhiganov, N. I., Tereshchenkova, V. F., Serebryakova,

M.V., Dunaevsky, Y. E., Belozersky, M. A., and Elpidina

E. N. (2025) Identification and localization of the set

of serine peptidases and their homologs in the lar-

val midgut of Tenebrio molitor L., Insect Mol. Biol.,

[Article submitted].

38. Vinokurov, K. S., Elpidina, E. N., Oppert, B.,

Prabhakar, S., Zhuzhikov, D. P., Dunaevsky, Y. E.,

and Belozersky, M. A. (2006) Diversity of digestive

proteinases in Tenebrio molitor (Coleoptera: Tene-

brionidae) larvae, Comp. Biochem. Physiol. B Bio-

chem. Mol. Biol., 145, 126-137, https://doi.org/10.1016/

j.cbpb.2006.05.005.

39. Perkin, L. C., Elpidina, E. N., and Oppert, B. (2017)

RNA interference and dietary inhibitors induce a

similar compensation response in Tribolium cas-

taneum larvae, Insect Mol. Biol., 26, 35-45, https://

doi.org/10.1111/imb.12269.

Publisher’s Note. Pleiades Publishing remains

neutral with regard to jurisdictional claims in published

maps and institutional affiliations. AI tools may have

been used in the translation or editing of this article.