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JOP. J Pancreas (Online) 2003; 4(2):83-88.

From Acute to Chronic Pancreatitis: The Role of Mutations in the Pancreatic

Secretory Trypsin Inhibitor Gene

Masahiko Hirota, Kinuko Kuwata, Masaki Ohmuraya, Michio Ogawa

Department of Surgery II, Kumamoto University Medical School. Honjo,

Kumamoto, Japan

Summary

Pancreatic secretory trypsin inhibitor (PSTI) is a potent natural inhibitor

of trypsin. We proposed the hypothesis that, if the function of the PSTI is

impaired by its genetic mutation, trypsin may easily promote autodigestion

causing pancreatitis and we performed a mutational analysis of the PSTI gene

in patients with pancreatitis. Two exonic mutations (N34S and R67C) were

thought to be associated with a predisposition to pancreatitis. The N34S

mutation was co-segregated with two intronic mutations, IVS1-37T>C and

IVS3-69insTTTT. Although we analyzed the function of the recombinant N34S

protein, we could not demonstrate the loss of function of this protein.

Intronic mutations, rather than N34S itself (IVS1-37T>C + N34S +

IVS3-69insTTTT complex), may be associated with the decreased function of

the PSTI. Alternatively, increased digestion of N34S in vivo may be

applicable. As for R67C, the conformational alteration of the protein by

forming intra-molecular or inter-molecular disulfide bonds with 67Cys was

strongly suggested. These results, along with the brand-new findings in PSTI

knockout mice, suggest that the genetic mutation of the PSTI is one of the

important mechanisms for predisposition to pancreatitis by lowering the

trypsin inhibitory function.

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Introduction

Inappropriate activation of trypsinogen within the pancreas leads to the

development of pancreatitis. Once trypsin is activated, it is capable of

activating many other digestive proenzymes. These activated pancreatic

enzymes further enhance autodigestion of the pancreas. Trypsin also

activates cells via the trypsin receptor. The trypsin receptor has recently

become known as one of the protease activated receptors, namely PAR-2. Both

acinar cells and duct cells express abundant PAR-2 [1].

Trypsin activity in the pancreas is mainly controlled by the pancreatic

secretory trypsin inhibitor (PSTI), which is also known as serine protease

inhibitor Kazal type 1 (SPINK1). The PSTI is synthesized in the acinar cells

of the pancreas, acts as a potent natural inhibitor of trypsin in order to

prevent the occurrence of pancreatitis. When trypsinogen is activated into

trypsin in the pancreas, the PSTI immediately binds to trypsin to prevent

further activation of pancreatic enzymes. The PSTI also blocks the further

activation of pancreatic cells via the trypsin receptor, PAR-2 (Figure 1).

Figure 1. Activation pathways of proenzymes and PAR-2 by trypsin. Once

trypsin is activated, it is capable of activating many other digestive

proenzymes. Trypsin also activates pancreatic and inflammatory cells via

PAR-2. The trypsin activity in the pancreas is mainly controlled by PSTI.

When trypsinogen is activated into trypsin in the pancreas, PSTI binds

immediately to trypsin to prevent further activation of pancreatic enzymes.

(PAR: protease activated receptors; PSTI: pancreatic secretory trypsin

inhibitor)

Several gene mutations in trypsinogen have been identified and are presumed

to be pathogenic in patients with hereditary pancreatitis through the

enhancement of intrapancreatic trypsin activity. The mutations lead to an

80% likelihood of developing pancreatitis. Although gene mutations in

trypsinogen have been identified and are presumed to be pathogenic in

patients with hereditary pancreatitis, no causative gene mutation was found

in about 50% of the patients. Subsequently, we proposed the hypothesis that,

if the function of the PSTI is impaired by its genetic mutation, trypsin may

easily promote autodigestion causing acute or chronic pancreatitis. Mutation

of the PSTI gene may promote a predisposition to pancreatitis, by lowering

the function of inhibiting trypsin activity. Five independent groups,

including ours, started at approximately the same time and reported the

mutational analysis of the PSTI gene in patients with pancreatitis [2, 3, 4,

5, 6, 7].

Mutational Analysis of the PSTI Gene in Familial and Juvenile Pancreatitis

in Japan

All 4 exons of the PSTI gene and their flanking intronic regions were

sequenced for 37 familial pancreatitis patients (24 families), 15 juvenile

pancreatitis patients, 22 sporadic pancreatitis patients (15 acute and 7

chronic) and 33 healthy volunteers.

Three types of exonic mutations in the PSTI gene were observed. N34S was

found in 6 familial pancreatitis patients (3 families) and 1 juvenile

pancreatitis patient, and R67C was found in one familial pancreatitis

patient and in one juvenile pancreatitis patient. It should be noted that

the N34S mutation was co-segregated with two intronic mutations,

specifically IVS1-37T>C and IVS3-69insTTTT (Table 1). The same set of

mutations (N34S + IVS1-37T>C + IVS3-69insTTTT) observed in other countries

was also observed in Japanese familial and juvenile pancreatitis patients.

Table 1. Summary of mutational analysis of PSTI in Japan.

Mutation

Familial

Pancreatitis

(n=74)

Juvenile

Pancreatitis

(n=30)

Sporadic

Pancreatitis

(n=44)

Healthy

Volunteer

(n=66)

IVS1-37T>C

8 (10.8%)

1 (3.4%)

0

0

Exon 3: N34S

8 (10.8%)

1 (3.4%)

0

0

IVS3-69insTTTT

8 (10.8%)

1 (3.4%)

0

0

Exon 4: R67C

1 (1.4%)

1 (3.4%)

0

0

Exon 4: 272C>T

2 (2.7%)

4 (13.3%)

6 (13.4%)

5 (7.6%)

PSTI: pancreatic secretory trypsin inhibitor

There is considerable support for the idea that the N34S mutation leads to

the development of pancreatitis: a) based on theoretical computer analysis

(Chou-Fosman and Robson-Garnier secondary structure prediction), it appears

that the N34S mutation may affect the conformation of the nearby active site

and then diminish the biological activity of PSTI [8]; the frequency of

the N34S mutation in pancreatitis patients was considerably higher than that

in non-pancreatitis subjects; c) the rate of association of pancreatitis in

subjects with the homozygous N34S mutation was assumed to be high based on

the data collected from recent reports (98%, 49/50). This high rate of

association of pancreatitis in homozygous N34S subjects suggests that this

mutation may be a recessive inherited trait.

The R67C mutation has not previously been discussed in reports from other

countries. Hence, R67C may be a uniquely Japanese mutation. The mature PSTI

protein has been reported to contain three intra-chain disulfide bonds:

32C-61C, 39C-58C and 47C-79C. The R67C mutation potentially forms a novel

disulfide bridge between 67Cys and any of the other Cys residues. 67Cys may

also form an intermolecular disulfide bond, such as PSTI homodimer or

PSTI-albumin complex (Figure 2). Alternatively, 67Cys may easily be

oxidized, producing a modified molecular form or causing the destruction of

acinar cells through endoplasmic reticulum stress.

Figure 2. Predicted molecular forms of R67C.

We also found a 272C>T mutation in the 3' untranslated region of exon 4 in 1

patient with familial pancreatitis, 4 patients with juvenile pancreatitis, 3

patients with sporadic acute pancreatitis and 3 patients with sporadic

chronic pancreatitis. This mutation, however, has been reported with high

frequency even in healthy volunteers and apparently indicates a normal

polymorphism (Table 1).

Functional Analysis of Recombinant PSTI Proteins with Amino Acid

Substitution

We hypothesized that mutation of the PSTI gene may promote predisposition to

pancreatitis, possibly by lowering the function of inhibiting trypsin

activity. Based on the hypothesis, we performed a biochemical analysis of

recombinant PSTI protein.

Trypsin inhibitory activity of recombinant protein was analyzed using human

and bovine trypsin [9]. The activity of the PSTI protein with a point

mutation of the most common type, N34S, was compared to that of the wild

type. The function of the N34S PSTI remained unchanged under both normal

alkali and acidic conditions as compared to the wild type PSTI (Figure 3).

Calcium concentration did not affect the activity of recombinant PSTI.

Trypsin susceptibility of the N34S protein did not increase either.

Figure 3. Inhibitory activity of recombinant PSTI proteins for human

trypsin. The function of N34S PSTI (red circles) remained unchanged as

compared to the wild type PSTI (blue squares). (PSTI: pancreatic secretory

trypsin inhibitor)

The interaction of recombinant N34S with human and bovine trypsin was also

analyzed by using a surface-plasmon-resonance (SPR) biosensor technique

[10]. The binding kinetics of the N34S PSTI protein to trypsin were compared

to those of the wild type. The binding kinetics of the N34S PSTI did not

decrease as compared to the wild type PSTI (Figure 4). These results along

with enzymatic functional analysis suggest that other mechanisms than the

conformational change of the PSTI by amino acid substitution may possibly

underlie the predisposition to pancreatitis in patients with N34S. Enhanced

digestion of N34S by enzymes other than trypsin and abnormal splicing may be

applicable.

Figure 4. Binding affinity of recombinant PSTI proteins to human trypsin.

The interaction of recombinant N34S with human trypsin was analyzed by using

an SPR biosensor technique. The binding kinetics of N34S PSTI did not

decrease as compared to the wild type PSTI. (PSTI: pancreatic secretory

trypsin inhibitor; SPR: surface-plasmon-resonance)

N34S is usually associated with two intronic mutations (i.e., IVS1-37T>C and

IVS3-69insTTTT). Mutations in the intronic polypyrimidine tract, such as

IVS3-69insTTTT which converts consecutive T5 to a T9 structure, sometimes

result in a splicing abnormality. Hence, intronic mutations rather than N34S

itself may be associated with the decreased function of the PSTI. The

failure to exhibit function loss in the recombinant N34S PSTI protein

supports this possibility.

As for R67C, there are many isoforms produced in the recombinant protein

producing system, as suggested above. All these isoforms of R67C recombinant

PSTI protein lost their reactivities with the anti-PSTI (wild type)

antibody, suggesting the massive conformational alterations. As a result of

the above-mentioned reasons, we could not purify recombinant R67C PSTI. R67C

is possibly associated with the predisposition to pancreatitis.

Future Perspectives

Genetic mutations in the PSTI gene seem to promote a predisposition to

pancreatitis, possibly by lowering the threshold for pancreatitis (Figure

5). To confirm the significance of the PSTI mutation, we are planning the

following projects: a) transcriptional and translational analysis of the

N34S mutation; processing analysis of R67C; c) analysis of PSTI-knockout

mice.

Figure 5. Intrapancreatic balance of trypsin and PSTI. Genetic mutations in

the PSTI gene seem to promote a predisposition to pancreatitis, possibly by

lowering the threshold for pancreatitis, as shown in the lowest situation.

(PSTI: pancreatic secretory trypsin inhibitor)

Among these projects, we have recently succeeded in generating PSTI-knockout

mice [11]. As to the heterozygous knockout mice, there was no alteration in

the macroscopic and microscopic views of the pancreas nor was there any sign

of pancreatitis. On the other hand, in the homozygous knockout mice of the

PSTI gene, the pancreas had disappeared. There are two possibilities which

may explain that phenomenon: a) failure of the pancreas to develop;

autolysis of the pancreas. Because we found necrotic remnants of the

pancreatic acinar cells in some siblings, the latter possibility may be

applicable. These results also support the significance of the PSTI

mutation.

Conclusion

Two exonic mutations (N34S and R67C) were thought to be associated with the

predisposition to pancreatitis. The N34S mutation was co-segregated with two

intronic mutations, IVS1-37T>C and IVS3-69insTTTT. Although we analyzed the

function of recombinant N34S protein, we could not demonstrate the

loss-of-function of this protein. Intronic mutations, rather than N34S

itself (IVS1-37T>C + N34S + IVS3-69insTTTT complex), may be associated with

the decreased function of the PSTI. Alternatively, increased digestion by

enzymes other than trypsin may be applicable. As for R67C, the

conformational alteration of the protein was strongly suggested. These

results, along with the brand-new findings in PSTI knockout mice, suggest

that the genetic mutation of the PSTI is one of the important mechanisms for

predisposition to pancreatitis.

References

1.. Kimura Y, Hirota M, Inoue K, Kuwata K, Ohmuraya M, Maeda K, Ogawa M.

Activation of protease-activated receptor 2 signals contributes to the

cytokine production in acute pancreatitis. In: Ogawa M, Yamamoto T, Hirota

M, eds. The Biological Response to Planned and Unplanned Injuries: Cellular,

Molecular and Genetic Aspects. Amsterdam (NL): Elsevier, in press. [More

details]

2.. Ogawa M, Hirota M, Kuwata K. Genetic mutations in pancreatic secretory

trypsin inhibitor (PSTI) gene in patients with pancreatitis. Annual Meeting

of Research Committee for Intractable Pancreatic Diseases in Japan. Tokyo,

September 9th, 1999. Tokyo, Japan: Research Committee for Intractable

Pancreatic Diseases in Japan, 1999. [More details]

3.. Kuwata K, Hirota M, Sugita H, Kai M, Hayashi M, Nakamura M, et al.

Genetic mutations in exons 3 and 4 of the pancreatic secretory trypsin

inhibitor in patients with pancreatitis. J Gastroenterol 2001; 36:612-8.

[More details]

4.. Witt H, Luck W, Hennies HC, Classen M, Kage A, Lass U, Landt O.

Mutations in the gene encoding the serine protease inhibitor, Kazal type I

are associated with chronic pancreatitis. Nature Genet 2000; 25:213-6. [More

details]

5.. Pfutzer RH, Barmada MM, Brunskill AP, Finch R, Hart PS, Neoptolemos J,

et al. SPINK/PSTI polymorphisms act as disease modifiers in familial and

idiopathic chronic pancreatitis. Gastroenterology 2000; 119:615-23. [More

details]

6.. Kaneko K, Nagasaki Y, Furukawa T, Mizutamari H, Sato A, Masamune A, et

al. Analysis of the human pancreatic secretory trypsin inhibitor (PSTI) gene

mutations in Japanese patients with chronic pancreatitis. J Hum Genet 2001;

46:293-7. [More details]

7.. Chen JM, Mercier B, Audrezet MP, Raguenes O, Quere I, Ferec C.

Mutations of the pancreatic secretory trypsin inhibitor (PSTI) gene in

idiopathic chronic pancreatitis. Gastroenterology 2001; 120:1061-3. [More

details]

8.. Kuwata K, Hirota M, Nishimori I, Otsuki M, Ogawa M. Mutational

analysis of the pancreatic secretory trypsin inhibitor (PSTI ) gene in

familial and juvenile pancreatitis in Japan. J Gastroenterol (in press).

[More details]

9.. Kuwata K, Hirota M, Shimizu H, Nakae M, Nishihara S, Takimoto A, et

al. Functional analysis of recombinant pancreatic secretory trypsin

inhibitor proteins with amino acid substitution. J Gastroenterol 2002;

37:928-34. [More details]

10.. Poiesi C, Albertini A, Ghielmi S, Cassani G, Corti A. Kinetic

analysis of TNF-alpha oligomer-monomer transition by surface plasmon

resonance and immunochemical methods. Cytokine 1993; 5:539-45. [More

details]

11.. Ohmuraya M, Hirokawa K, Araki M, Araki K, Hirota M, Ogawa M, Yamamura

K. Generation of pancreatic secretory trypsin inhibitor gene knockout mice.

In: Ogawa M, Yamamoto T, Hirota M, eds. The Biological Response to Planned

and Unplanned Injuries: Cellular, Molecular and Genetic Aspects. Amsterdam

(NL): Elsevier (in press). [More details]

Article in PDF format

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Keywords Acute Disease; Chromosome Disorders; Chronic Disease; Enzyme

Activators; Genetic Diseases, Inborn; Mutation; Pancreatitis; Serine

Proteinase Inhibitors; Surface Plasmon Resonance; Trypsin Inhibitor, Kazal

Pancreatic; Trypsin; Trypsin Inhibitors; Trypsinogen

Abbreviations PAR: protease activated receptors; PSTI: pancreatic secretory

trypsin inhibitor; SPINK1: serine protease inhibitor Kazal type 1; SPR:

surface-plasmon-resonance

Correspondence

Masahiko Hirota

Department of Surgery II

Kumamoto University Medical School

1-1-1 Honjo

Kumamoto 860-8556

Japan

Phone: +81-96-373.5212

Fax: +81-96-371.4378

E-mail address: mhirota@...

I hope this finds you and yours well

Mark E. Armstrong

casca@...

www.top5plus5.com

PAI NW Rep

ICQ #59196115

OK, GUYS PLEASE HELP!!

> Just to be brief...I jut now returned home from my EUS at the Lahey

> Clinic. After the procedure, I had written notes from the doctor

> (That I THOUGHT was so wonderful!!).....he said in the notes " I see

> minimal changes of chronic pancreatitis. I am not sure that I can

> attribute your pain to these changes. " HUH?????? This is the same

> doc that was talking about damage occurring on

> a " microscopic/cellular level " when I first went to the initial

> consult with him. I demanded that he speak to me right then and

> there (after the proceduree) because the notes he left had me

> wondering and then after talking to him literally in tears. He said

> I cannot diagnose CP because I need to see 7 signs and you only have

> 2 of the seven and these can be attributed to anyone's pancreas in

> someone your age. " I'm 46....so he now is saying that he won't give

> me a definitive dx of cp!! When asked about the possibility of SOD

> which I had mentioned to him at the first consult and as some of you

> may recall...at the time he poo poo'd the notion and said it's

> usually used when doctors don't know what else to call it.....I

> asked about an ERCP w/manometry...and he said we don't do that

> here. So, when pushed by me...he finally said, I don't believe that

> you have cp because your pain cannot be attributed to the minimal

> changes I saw in your pancreas today...then he flip flopped and

> said " I can't rule it out either....but in my opinion, you don't

> have it because we would have been seeing more damage. " Soooooo, he

> is now in the catetory of the DINKS!!! So, what do I do now? He

> put on the release slip - return to work tomorrow and the one that

> really got me!! RESUME ALCOHOL tomorrow!! I guess what I need here

> is some BIG TIME reassurance and advice from you guys....PLEASE

> HELP!! I mean, I know of some of you that CP wasnt' dx'd UNTIL the

> performed an ERCP with manometry due to SOD!!! I'm about to freak

> here!!! Please write back....I'm sooo confused right now!!! I KNOW

> WHAT I HAVE!!!

> thanks and sorry for this crazy message, but I just don't know what

> to do now!!! I can't return to work...but if he isn't willing to dx

> CP - I can't apply for SSID w/o a dx either right???

> thanks

>

>

>

>

>

>

>

>

[Guest guest]

MARK!

Woooohooooo and a big Whao Bundy!

Man... that's some good reading man! THANX.. Hey.. How the heck does

one GET on a study like this, as one of the group WITH of course...

I'd LOVE to help to further the research and undertsnading of this

desease...

Very interesting read.

[Guest guest]

I know Mark....sorry for the " tone " of my email. I'm sure most of

you have been down that road if not once, many times before. I

guess I was just shocked because though I didn't expect the EUS to

actually show much (I'm only about 6 months into this) I didn't

expect his flip-flopping opinion. When I went to the initial

consult with him I was SO pleased because he made remarks to me

about how ANY disease starts at a " microscopic, cellular level FIRST

before manifesting in yadda, yadda, yadda.... " I just can't tell

you how profoundly sad I am right now...and that kind of shocks me

too (my own disturbance by this that is -I've heard so much from you

guys that I didn't think I'd be this upset but here it is the next

day and I'm still feeling sad.) I really thought I had been lucky in

finding a good GI doc...

Thanks for your immediate reply and support though Mark...I

appreciate that alot.

[Guest guest]

You just gave me one of the first smiles I've had all day Mark!!

Tone away at you!! LOL!!

[Guest guest]

,

have you thought about going to another doctor for a second opinion. You

are entitled to have one and it certainly sounds like you need one. I am

so sorry that your current doctor is waffling on diagnosing you.

Kimber

--

Kimber

Vallejo, CA

hominid2@...

Note: All advice given is personal opinion, not equal to that of a licensed

physician or health care professional.