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Complete primary structure of lamb preprochymosin deduced from cDNA

Authors:
Joze Pungercar at Jožef Stefan Institute
Joze Pungercar
Borut Štrukelj
Borut Štrukelj
Borut Štrukelj
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Franc Gubenšek
Franc Gubenšek
Franc Gubenšek
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Vito Turk at Jožef Stefan Institute
Vito Turk
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4602
Nucleic
Acids
Research,
Vol.
18,
No.
15
Complete
primary
structure
of
lamb
preprochymosin
deduced
from
cDNA
Joze
Pungercar,
Borut
Strukelj,
Franc
Gubensek,
Vito
Turk
and
Igor
Kregar
Department
of
Biochemistry,
Jocef
Stefan
Institute,
Jamova
39,
61111
Ljubljana,
Yugoslavia
Submitted
June
27,
1990
EMBL
accession
no.
X53037
A
cDNA
coding
for
lamb
preprochymosin
was
isolated
from
an
abomasum
cDNA
library
successively
screened
wit
two
oligonucleotide
probes
corresponding
to
the
5'-
and
3'-ends
of
the
coding
region
for
calf
prochymosin
(1-3).
Similar
to
the
situation
for
the
calf
enzyme,
a
16
amino
acid
residue
signal
peptide
(underlined)
is
followed
by
42
residues
of
the
proenzyme
region.
The
mature
lamb
chymosin
begins
with
glycine
at
position
59.
The
coding
nucleotide
and
deduced
amino
acid
sequences
of
lamb
preprochymosin
show
about
95%
and
94%
similarity
to
calf
preprochymosin,
respectively.
Both
aspartic
acid
residues
in
the
active
site
at
positions
92 and
274
are
conserved.
Lamb
chymosin
described
here
can
be
denoted
as
the
B
form
because
of
the
presence
of
glycine
at
position
302
(cf.
4).
ACKNOWLEDGEMENTS
We
thank
Roman
Jerala
for
synthesis
of
the
C-terminal
probe.
This
work
was
supported
by
the
grants
of
te
Research
Community
of
Slovenia,
Federal
Secretariat
for
Development
and
Krka
Pharmaceutical
Works.
REFERENCES
1.
Hairris,T.J.R.,
Lowe,P.A.,
Lyons,A.,
Thomas,P.G.,
Eaton,M.A.W.,
Milfican,T.A.,
Patel,T.P.,
Bose,C.C.,
Carey,N.H. and
Doel,M.T.
(1982)
Nucl.
Acids
Res.
10,
2177-2187.
2.
Moir,D.,
Mao,J.-I.,
Schumm,J.W.,
Vovis,G.F.,
Alford,B.L.
and
Taunton-
Rigby,A.
(1982)
Gene
19,
127-138.
3.
Nishimori,K.,
Kawaguchi,Y.,
Hidaka,M.,
Uozumi,T.
and
Beppu,T.
(1982)
J.
Biochem.
91,
1085-1088.
4.
Foltmann,B.,
Pedersen,V.B.,
Kauffman,D.
and
Wybrandt,G.
(1979)
J.
Biol.
Chem.
254,
8447-8456.
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360
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1260
1305
k./
1990
Oxford
University
Press

Supplementary resource (1)

Lamb mRNA for preprochymosin (EC 3.4.23.4)
Nucleotide Sequence
May 1990
F. Gubensek
... A single aspartic proteinase zymogen, lamb prochymosin, has been found in which the conserved basic residue at position 36p is replaced by an acidic residue, Glu, as shown by cDNA cloning [9]. This suggests that either other sequence elements of the lamb propeptide compensate for the proposed folding role of Lys36p or a basic residue at position 36p is not essential for folding and processing. ...
... Genomic DNA was isolated from spleens of lamb (Ovis aries), mouflon (Ovis musimon) and goat (Capra hircus) using a standard procedure [10]. Primers for PCR were constructed according to the lamb prochymosin cDNA sequence [9] and the structure of the calf prochymosin gene [11] to amplify the presumed exon 2 of the homologous genes. Oligonucleotides were designed taking into consideration the rules for selection of PCR primers [12]. ...
... The coding regions of lamb and calf prochymosin cDNAs [9,15] were separately inserted into the phagemid pSE-LECT-1 (Promega) and mutated in vitro at position 36p according to the manufacturer's instructions. The sense mutagenic CCM (5 H -CATCAGCAGCGAGTACTCCGG-3 H ) and LCM oligonucleotides (5 H -CGTCAGCAGCAAGTAC-TCCGG-3 H ) introduced point mutations Lys36p3Glu and Glu36p3Lys in calf and lamb prochymosins, respectively. ...
A basic residue at position 36p of the propeptide is not essential for the correct folding and subsequent autocatalytic activation of prochymosin
Article
  • Dec 2001
  • Eur J Biochem
Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the active-site aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7. All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms. Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A [Richter, C., Tanaka, T., Koseki, T. & Yada, R.Y. (1999) Eur. J. Biochem.261, 746–752] can be reconciled with those for prochymosin. Lys/Arg36p is involved in stabilizing the propeptide–enzyme interaction, along with residues nearer the N-terminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former.
View
... From 1980 to 2019, recombinant chymosins of sev eral mammals were produced and characterized: sheep [11,12], goat [13,14], camel (dromedary) [15,16], yak [17,18], buffalo [19], and alpaca [20]. Examination of biochemical and technological properties of the produced enzymes revealed that the recombinant camel chymosin had higher milk clotting activity and specificity than the bovine chymosin (milk clotting and non specific prote olytic activity ratio), but demonstrated higher thermal stability (TS) [16,21,22]. ...
Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties
Article
Full-text available
  • Jul 2020
For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichia coli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis-Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s-1 and 2.27± 0.10 µm M-1·s-1, respectively.
View
... The primary structures of animal rennets from other ruminants are very similar to that of bovine chymosin. Sequencing of the cDNA fragments of lamb (Pungercar et al. 1990), buffalo (Vallejo et al. 2008) and camel (Kappeler et al. 2006) preprochymosins revealed similarities of The DTG residues that are critical for catalytic activity are highlighted in yellow. The disulphide bridges are highlighted in red 95%, 97.9%, and 87%, respectively to that of bovine preprochymosin. ...
Progress in the field of aspartic proteinases in cheese manufacturing: Structures, functions, catalytic mechanism, inhibition, and engineering
Article
  • Nov 2013
Aspartic proteinases are an important class of proteinases which are widely used as milk-coagulating agents in industrial cheese production. They are available from a wide range of sources including mammals, plants, and microorganisms. Various attempts have been made in order to get insights into enzyme structure/function relationships for designing improved biocatalysts. This review provides an overview of historical background and recent achievements on the classification and structural characteristics of such enzymes as related to their functional properties, mechanism of catalysis, pH, and temperature dependence, substrate specificities, mechanism of inhibition, enzyme engineering, and technological applications with the focus on cheese manufacturing.
View
... The carbohydrate components are connected to asparagine (Baudys et al., 1988). Pungercar et al. (1990) have reported 94% homology of lamb prochymosin with calf prochymosin. Bovine pepsinogen consists of 362 ± 2 amino acids and has molecular weight (MW) 38.9 kDa (Chow and Kassell, 1968). ...
Characteristics of rennet and other enzymes from small ruminants used in cheese production
Article
Full-text available
  • Nov 2011
  • SMALL RUMINANT RES
Rennet from small ruminants is widely used in Mediterranean countries to produce various cheeses, including Protected Designation of Origin (PDO) cheeses. Usually, this rennet is produced by the cheesemakers and consequently, this artisanal product has some special characteristics, compared to other rennets. Apart from the proteolytic enzymes, this rennet type contains also lipolytic enzymes that contribute to the produced cheese special sensory characteristics. This review outlines the preparation procedures, the enzymatic characteristics as well as the physicochemical composition and microbiological quality of such rennet and also presents the use of other commercial enzyme preparations from lamb and kid in cheesemaking.
View
... Foltmann (1964) analyzed amino acids of purified chymosin and reported values that were almost in agreement with the amino acid composition for chymosin reported by earlier workers. Several workers have cloned and sequenced chymosin cDNA to completely deduce the nucleotide and amino acid sequence (Harris et al., 1982;Hidaka et al., 1986;Pungercar et al., 1990;Vega-Hernandez et al., 2004;Kumar et al., 2006). ...
Chymosin and other milk coagulants: Sources and biotechnological interventions
Article
Calf rennet, which consists of over 90% chymosin, is commonly used in cheese industries for the curdling of milk. Various animal, plant and microbial sources have been exploited as possible alternatives to calf rennet. The coagulating properties of the enzymatic preparations (coagulants) from these sources differ in terms of their physicochemical factors. The cheese industry has always sought out novel and stable enzyme sources, and recombinant chymosin has been found to be an effective alternative since it possesses several advantages over plant and microbial milk-clotting enzymes. This paper reviews the use of various milk coagulants, especially animal coagulants, for cheese making. Advancements in genetic and protein engineering to produce recombinant chymosin are discussed in addition to evaluating its identity to the rennet available from natural sources.
View
The Structural Variety and Metabolism of Proteins
Chapter
  • Jan 1994
Individual eukaryote cells contain in the order of 104 different proteins, and each animal species contains an even greater number due to differences between the tissues of an individual and between the individuals themselves; furthermore, the protein spectrum changes during the course of development. The number of different proteins to be found in extant organisms may be as high as 1012. The description of this variety, its origin and biological significance is the most extensive theme in comparative biochemistry. This chapter will concern itself with the possibilities for structural variation and the general metabolism of proteins; further chapters will deal with comparative studies of individual proteins.
View
General and Molecular Aspects of Rennets
Chapter
  • Jan 1993
Cheesemaking and fermentation represent the first examples of applied biochemistry and biology. Whereas living microorganisms are used in fermentation processes, the clotting of milk for cheesemaking has always required soluble enzymes. The milk-clotting enzyme from the fourth stomach of the calf was one of the first enzymes of which purification was attempted, and Deschamps1 suggested the name chymosin, derived from the Greek word for gastric liquid ’chyme’. This designation was later used in continental European languages, whereas in English the name rennin, derived from rennet, was used.2 Misunderstandings often occurred between rennin and renin from the kidneys, and therefore the designation chymosin was recently adopted in English3 and it is now used in the recommended international enzyme nomenclature.4
View
Structure and Proposed AminoAcid Sequence of a Pepsin from Atlantic Cod (Gadus morhua)
Article
  • Jan 1998
The crystal structure of a pepsin from the gastric mucosa of Atlantic cod has been determined to 2.16 Å resolution. Data were collected on orthorhombic crystals with cell dimensions a = 35.98, b = 75.40 and c = 108.10 Å, on a FAST area-detector system. The phase problem was solved by the molecular-replacement method using porcine pepsin (PDB entry 5PEP) as a search model. The structure has been refined to a crystallographic R factor of 20.8% using all reflections between 8.0 and 2.16 Å, without prior knowedge of the primary sequence. The resulting crystal structure is very similar to the porcine enzyme, consisting of two domains with predominantly β-sheet structure in the same sequential positions as the enzyme from pig. In the course of the model building, 122 residues were substituted and two residues deleted from the starting model to give a polypeptide chain of 324 amino acids and a sequence identity of 57.7% with the pig pepsin. No carbohydrate residues were located. Sequence alignment with available aspartic proteinases, indicates that the fish enzyme seems to be more related to mammalian gastric pepsins than to the mammalian gastricsins and chymosins, lysosomal cathepsin D's and a pepsin from tuna fish. The amino-acid composition of the cod enzyme, however, is more in accordance with the cathepsin D's.
View
Aspartic Proteases Used in Cheese Making
Chapter
  • Jan 2007
The use of aspartic proteases (APs) in cheese manufacture is among the earliest applications of enzymes in food processing, dating back to approximately 6000 B.C. (Fox and McSweeney, 1999). Enzymatic milk coagulation is a two-phase process. In the first phase, APs hydrolyse the Phe 105-Met106 bond of bovine κ-casein splitting the protein molecule in two, yielding hydrophobic para-κ-casein and a hydrophilic part known as the macropeptide. The second phase consists of the coagulation of the casein micelles that have been destabilized by the proteolytic attack. Milk-clotting enzymes are obtained from mammals, plants and fungi. They can also be produced using recombinant DNA technology. Enzymes extracted from the fourth stomach (abomasum) of suckling calves (rennet) have traditionally been used as milk coagulants for cheese production. In addition to its chymosin content, conventional rennet also contains lower levels of pepsin A, the most representative peptidase of Family A1, characterized by its general proteolytic activity that makes it unsuitable for milk clotting (Harboe and Budtz, 1999). Plant and fungal milk coagulants present high levels of non-specific, heat-stable proteases the prolonged action of which cause bitterness in the cheese after a period of storage (Harboe and Budtz, 1999; Roserio et al., 2003). A world shortage of bovine rennet, due to the increased demand for cheese, encouraged the search for alternative milk coagulants. Research on fungal APs resulted in the production of enzymes that are inactivated at normal pasteurisation temperatures and contain low levels of non-specific proteases (Branner-Jorgensen et al., 1982; Yamashita et al., 1994; Aikawa et al., 2001). In 1988, chymosin produced by recombinant DNA technology was first introduced to the dairy industry for evaluation. A few years later, scientists at Genencor International were able to increase the production of chymosin in Aspergillus niger var. awamori to commercial levels (Dunn-Coleman et al., 1991). Presently, several recombinant chymosins such as Maxiren ® produced by DSM, and Chymogen ® produced by Christian Hansen, are available on the market. Recombinant chymosin preparations are very pure and have high milk-clotting activity. Chymosins from other mammalian species including lamb, kid goat, camel and buffalo calves are being considered as alternatives for milk clotting in the production of certain types of cheese (Mohanty et al., 1999; Elagamy, 2000; Rogelj et al., 2001; Vega-Hernández et al., 2004).
View
PsoP1, a Milk-Clotting Aspartic Peptidase from the Basidiomycete Fungus Piptoporus soloniensis
Article
  • Sep 2011
  • J AGR FOOD CHEM
The first enzyme of the basidiomycete Piptoporus soloniensis, a peptidase (PsoP1), was characterized after isolation from submerged cultures, purification by fractional precipitation, and preparative native-polyarylamide gel electrophoresis (PAGE). The native molecular mass of PsoP1 was 38 kDa with an isoelectric point of 3.9. Similar to chymosin from milk calves, PsoP1 showed a maximum milk-clotting activity (MCA) at 35-40 °C and was most stable at pH 6 and below 40 °C. The complete inhibition by pepstatin A identified this enzyme as an aspartic peptidase. Electrospray ionization-tandem MS showed an amino acid partial sequence that was more homologous to mammalian milk clotting peptidases than to the chymosin substitute from a fungal species, such as the Zygomycete Mucor miehei. According to sodium dodecyl sulfate-PAGE patterns, the peptidase cleaved κ-casein in a way similar to chymosin and hydrolyzed β-casein slowly, as it would be expected from an efficient chymosin substitute.
View
The primary Structure of calf chymosin
Article
Full-text available
  • Oct 1979
The complete amino acid sequence of calf chymosin (rennin) (EC 3.4.23.4) has been determined. The sequence consists of a single peptide chain of 323 amino acid residues. The primary structure of the precursor part of calf prochymosin was published previously (Pedersen, V.B., and Foltmann, B. (1975) Eur. J. Biochem. 55, 95-103), thus we are now able to account for the total 365 amino acid residues of calf prochymosin. Comparison of the sequence of calf prochymosin with that of pig pepsinogen A (EC 3.4.23.1) shows extensive homology. In the precursor part of the sequence, 15 residues are located at identical positions, as compared to 189 identical residues in the respective enzymes. Furthermore comparison to Penicillium janthinellum acid proteinase (penicillopepsin) (EC 3.4.23.7) shows that 76 residues are common to this enzyme and to the two gastric proteinases. These homologies in sequence further suggest that the folding of the peptide chain in chymosin is very similar to that of other acid proteinases.
View
Molecular cloning and characterisation of double-straned cDNA coding for bovine chymosin
Article
  • Jul 1982
  • GENE
A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the Mr-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.
View
Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin
Article
  • May 1982
DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pstl site of pAT153 by G-C tailing. Clones containing sequences coding for prochymesin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymesin1. Two clones were identified which together contained a complete copy of prochymesin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin2 and shows that this protein has a mol.wt. of 40431 and chyemsin a mol.wt. of 35612. The sequence also indicates that prochyn is synthesised as a precursor melecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins3.
View
Nucleotide Sequence of Calf Prorennin cDNA Cloned in Escherichia coli
Article
  • Apr 1982
The nucleotide sequence of prorennin (prochymosin) cDNA cloned in E. coli was determined by the technique of Maxam and Gilbert. The longest prorennin cDNA insert in pTACR1 contained the putative signal sequence and the coding sequence for the peptide from the 1st amino acid, Ala (NH2 terminal), to the 296th, Ser, and the other clone pTACR9 contained the coding sequence from the 258th, Asp, to the 365th, Ile (COOH terminal), and the TGA termination codon followed by the 3'-untranslated region. Thus, the whole coding sequence for prorennin was obtained in the pair of pTACR1 and pTACR9.
View
  • Jan 1982
  • 2177-2187
  • T J R Hairris
  • P A Lowe
  • A Lyons
  • P G Thomas
  • M A W Eaton
  • T A Milfican
  • T P Patel
  • C C Bose
  • N H Carey
  • M T Doel
Hairris,T.J.R., Lowe,P.A., Lyons,A., Thomas,P.G., Eaton,M.A.W., Milfican,T.A., Patel,T.P., Bose,C.C., Carey,N.H. and Doel,M.T. (1982) Nucl. Acids Res. 10, 2177-2187.