ELSEVIER Biochimica et Biophysica Acta 1253 (1995) 9-12

BB Biochi ~mic~a et Biophysica A~ta

Rapid Report

Processing and activation of recombinant mouse mastocytoma histidine decarboxylase in the particulate fraction of Sf9 cells by porcine

pancreatic elastase

Satoshi Tanaka, Tetsuya Fukui, Jun Yamamoto, Yoh-ichiro Shima, Toshiaki Kume, Makoto Ohgo, Atsushi Ichikawa *

Department of Physiological Chemisto', Facul(v of Pharmaceutical Sciences, Kyoto UniversiO', Sakyo-ku, Kyoto 606, Japan

Received 5 April 1995; revised 10 August 1995; accepted 16 August 1995

Abstract

Mature 53 kDa histidine decarboxylase (HDC) peptide is produced from a precursor 74 kDa peptide. The mechanism of specific cleavage by processing enzyme is unknown. Using the recombinant mouse 74 kDa HDC, we found that porcine pancreatic elastase specifically converted the inactive 74 kDa HDC to its active form of 53 kDa HDC.

Keywords: Histidine decarboxylase; Sf9 cell; Mastocytoma; Pancreatic elastase; (Mouse); (Porcine)

L-Histidine decarboxy]lase (HDC: EC 4.1.1.22) is the only enzyme that forms histamine, which plays an impor- tant role in diverse biological functions among mammals [1-4]. Thus, the clarification of the mechanism underlying the regulation of the enzyme is of great importance to understand such various functions. For this purpose, HDC has been purified and ch~tracterized from the soluble frac- tions of various sources [5-8]. The purified HDC com- prises a homodimer, the molecular mass of the enzyme subunit being 53-55 kDa. On the other hand, cloning of the cDNAs for the enzyme [9-11] has indicated that the initial translation products are all around 74 kDa. How- ever, no evidence for the presence of the 74 kDa species nor its post-translational processing resulting in the forma- tion of the 53 kDa species has been identified in any mammalian tissues. Recently, we have demonstrated that the recombinant mouse mastocytoma 74 kDa HDC (74K- rHDC) expressed in Sf9 cells is located in the particulate fraction of the cells, whereas the C-terminal deleted, re-

Abbreviations: HDC, L-histidine decarboxylase; PPE, porcine pancre- atic elastase; PCR, polymerase chain reaction; cDNA, complementary DNA; GST, glutathione S-transferase.

* Corresponding author. Tel.: +81 75 7534527; fax: +81 75 7534557.

0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 01 67-4838(95)001 85-9

combinant 54 kDa peptide species (54K-rHDC) is located in the soluble fraction [12].

Proteinases have been known to convert nascent or inactive proteins into mature or active polypeptides [13], and to change the intracellular localization of the mature proteins so that they can express their biological functions [14]. Therefore, it is likely that the 74 kDa HDC is converted into more active 53 kDa HDC species by the action of proteinase(s). To clarify the molecular mecha- nisms of the conversion of HDC species, it is indispens- able to examine the effect of exogenous proteinase on it. Thus, we have investigated whether 74K-rHDC is specifi- cally cleaved to form 53K-rHDC by proteinase(s) in vitro.

74K-rHDC was expressed in Sf9 cells and found to be mostly present in the particulate fraction of centrifugation at 100 000 × g and practically inactive (about 60 nmol /min per mg vs. 800 nmol/min per mg of the purified HDC [6]) [12]. Since the treatment of the particulate 74K-rHDC with 1% Triton X-100, 0.1% Tween 20, 1% cholate-Na ÷ or 10 mM CHAPS did not cause the release of immunoreactive 53K-rHDC [12], the slight HDC activity in the particulate fraction may be derived from the 74K-rHDC itself. An aliquot of the particulate fraction was incubated with vari- ous proteinases such as porcine pancreatic elastase (PPE, Sigma, St. Louis, MO) for 10 min at 37°C. HDC activity

10 S. Tanaka et al. / Biochimica et Biophysica Acta 1253 (1995) 9-12

None

Trypsin (10 Hg/ml)

Chymotrypsin (10 pg/ml)

Papain (50 ~g/ml)

Collagenase (10 Hg/ml)

PPE (6 ~g/ml)

HLE (10 ~g/ml) I I I

20 40 60

HDC Activity (pmol/min)

Fig. 1. Effect of various proteinases on HDC activity in the particulate recombinant mouse mastocytoma HDC (74K-rHDC). After Sf9 cells infected with recombinant virus were harvested, they were washed and suspended in 50 mM potassium phosphate buffer (pH 6.8), containing 0.2 mM dithiothreitol, 0.01 mM pyridoxal 5'-phosphate, 2% polyethylene glycol #300, and disrupted by freezing in liquid nitorogen and thawing at 37°C. The suspension was then centrifuged at 100000X g for 20 min at 4°C, and the particulate fraction was washed and resuspended in the same buffer. An aliquot of the particulate fraction (2.3 mg protein/ml) was incubated for 10 min at 37°C with the indicated concentrations of trypsin, chymotrypsin, papain, collagenase, porcine pancreatic elastase (PPE), or human leukocyte elastase (HLE). An aliquot of each reaction mixture was assayed for enzyme activity. Values represent the mean + S.E. (n = 6).

was assayed by o-phthalaldehyde method [15] as described previously [6]. As shown in Fig. 1, the total HDC activity in the reaction mixture increased about 6-fold by PPE treatment. However, the PPE-caused HDC activity was not much more by a further incubation (data not shown). Contrast to PPE, human leukocyte elastase (HLE, Sigma, St. Louis, MO), which has a different spectrum of sub- strate specificity from PPE [16,17], did not stimulate the

activity of the particulate fraction. PPE-caused HDC activ- ity was completely abolished by adding elastatinal (10 /zg /ml ) , a specific elastase inhibitor [18], and phenyl- methylsulfonyl fluoride (1 mM), a serine proteinase in- hibitor (data not shown). However, PPE was insensitive to the activity of the mature HDC purified from mouse mastocytoma cells [6]. Although collagenase and papain slightly increased the total activity, the addition of a larger amount of proteinase or the extension of incubation time caused almost complete inactivation of HDC, indicating that the increase in activity caused by co l lagenase /papa in is distinct from that caused by PPE. This result means PPE is specific for increase of the HDC activity in the particu- late fraction containing 74K-rHDC.

After centrifugation of reaction mixture containing PPE-treated particulate fraction at 100000 X g for 20 min, over 90% of PPE-caused HDC activity was recovered in the supernatant fraction (data not shown). The molecular species of PPE-caused HDC activity in the supernatant fraction was estimated by immunoblotting using specific antibody against GST-HDC fusion protein. Samples of both the supernatant and particulate fractions, obtained at 100000 X g centrifugation, were subjected to SDS-PAGE on a slab gel, and the separated proteins were then trans- ferred electrophoretically to a PVDF membranes. The membrane was incubated with an anti-GST-HDC antibody, which had been raised against the GST-HDC fusion pro- tein. The GST-HDC fusion protein was produced in E. col i strain HB101 transformed with the expression vector pGEX-2T (Pharmacia) being inserted a 630 bp fragment of mouse HDC cDNA (Nucleotide 1-630), and was purified by glutathione-coupled Sepharose 4B affinity column chro- matography. The immunoblot analysis (Fig. 2) showed that PPE treatment to the particulate fraction containing 74K-

competitor None

1 2 3 4 5

GST-HDC

7 8 9 10

94 67

43

30

20

<1

Fig. 2. Conversion of the particulate 74K-rHDC into soluble 53K-rHDC species by PPE. The PPE-treated particulate fraction prepared by the procedure described in the legend of Fig. 1 was centrifuged at 100000 X g for 20 min. Aliquots of the obtained supernatant and particulate fractions from PPE-treated 74K-rHDC species were immunoblotted using affinity-purified anti-HDC antibody (1:1000), with (right) or without (left) GST-fusion HDC (5 /~g/ml). Lanes 1 and 6, the supernatant without PPE, Lanes 2 and 7, the precipitate without PPE, Lanes 3 and 8, the supernatant with PPE, Lanes 4 and 9, the precipitate with PPE, lanes 5 and 10, purified mouse mastocytoma HDC (15 ng) [6]. 74K-rHDC and 53K-rHDC were indicated by the white and black arrow heads, respectively.

S. Tanaka et al. / Biochimica et Biophysica Acta 1253 (1995) 9-12 11

80

i- >~ 40

-

< 20

g 3: 0

| i i i t

210 140 67 24

J J J J , ~ PPE-Treatmen

" ' , - - -0-- - Purified HDC

11 13 15 17 19

Fraction No.

94--> 6 7 - > 4 3 - >

3 0 - >

B Fraction No.

11 13 15 17 19

<]

Fig. 3. Molecular masses of PPE-released HDC species. The supematant from PPE-treated 74K-rHDC or the purified mouse mastocytoma HDC [6] was separately applied to a Superose 12 HR 10/30 (Pharmacia, Uppsala) column eqilibrated wita 50 mM potassium phosphate (pH 6.8) containing 0.2 M NaCI, 0.2 mM dithiothreitol, 0.01 mM pyridoxal 5' phosphate, 2% polyethylene glycol #300. Aliquots of the fractions were assayed for the enzyme activity (A) or immunoblotted on SDS-PAGE (B) to determine the size of the HDC peptide. The molecular mass markers were catalase (210 kDa), lactate dehydrogenase (140 kDa), malate dehy- drogenase (67 kDa), and c~-chymotrypsinogen (24 kDa). 53K-rHDC was indicated by the arrow head.

rHDC resulted in the formation of major 53K-rHDC and minor 55K-rHDC in the soluble fraction, and the decrease of particulate 74K-rHDC. Two other bands, corresponding to 81 and 250 kDa, were also visible even in the presence of excess GST-HDC fusion protein. Therefore, these two bands seemed to be non-specific products that reacted with the antibody. Furthermore, the soluble fraction of collage- nase- or papain-treated particulate recombinant HDC was demonstrated to form about 60 kDa band but no 53-55 kDa bands (data not shown).

Next, we estimated the molecular mass of the HDC species converted by PPE to the soluble form from the particulate recombinant 74 kDa HDC by means of Super- ose 12 gel-filtration column chromatography (Fig. 3A). The molecular mass of the PPE-released HDC species, determined by the HDC activity, was about 110 kDa, which is identical to that of purified mastocytoma HDC. On the immunoblots, the amount of the immunoreactive 53 kDa HDC species, but not that of the other visible

immunoreactive bands was proportional to the HDC activ- ity (Fig. 3B). These results suggested that PPE-released recombinant HDC species is a homodimer consisting of two identical 53 kDa HDC subunits like the mature mouse HDC [6].

The catalytic activity of PPE-released HDC was deter- mined and compared with the mature HDC purified from mouse mastocytoma cells [6]. The isoelectric point was 5.4 by chromatofocusing on a Mono P HR5/20 column, the optimal pH was 6.6, and the K m value for L-histidine was 0.28 mM (data not shown). Each value was similar to respective one of the purified mouse mastocytoma HDC [6]. The result indicates that PPE-released recombinant HDC species has the same catalytic properties as the mature mouse HDC species, and the catalytic activity does not seem to be affected by PPE treatment.

In this study we revealed that PPE is a model for the putative processing enzyme converting the 74 kDa HDC in the particulate fraction to the active mature HDC compris- ing a homodimer of a 53 kDa subunit in the soluble fraction. The immunoblot analysis with an anti-GST-HDC peptide antibody showed that the antibody recognizes re- combinant 74 kDa HDC as well as its PPE-released HDC. Since the fusion protein was comprised Met~-Ile 21° amino acids of mouse HDC combined to the C-terminus of GST, it is likely that proteolytic cleavage of 74K-rHDC would result in the formation of 53K-rHDC comprising of N- terminal polypeptide from 74K-rHDC peptide. Interest- ingly, a sequence of 47VGlu-Ala-Ala-Asn-Leu-Val-Leu- 483Ser [11], which contains considerably many putative cleaving sites of PPE [16], is present in the cDNA-derived amino-acid sequence of 74K-rHDC. Cleavage of one of these sites would result in the formation of around 53K- rHDC by deleting about 21 kDa C-terminal region of 74K-rHDC. Regrettably, insufficient 53K-rHDC is re- leased by PPE for us to precisely determine the cleavage site by C-terminal sequencing.

It is quite possible that the C-terminal 21 kDa region is involved in the interaction of the 74K-rHDC with cellular membranes and intracellular organelles, or in the aggrega- tion of each other. However, the C-terminal region is not so hydrophobic and contains several cysteine residues, suggesting the anchorage of the region to cellular structure through disulfide bonds [11,12]. Recently we have found the immunoreactive 74 kDa HDC species in the particulate fraction of mouse stomach and the PPE-induced release of immunoreactive mature HDC consisting a homodimer of a 53 kDa subunit (manuscript in preparation). Therefore, cleavage by proteinase(s) of the C-terminal region of the translated 74 kDa HDC may be required to discharge active form of a matured HDC consisting a homodimer of a 53 kDa subunit in the mouse. With regards to pro- teinases, however, the known etastase-like proteinases, e.g. PPE, HLE, and cathepsin G, are all secreted ones [16]. Thus, further study is necessary to identify the endoge- nously active proteinase(s) instead of PPE.

12 S. Tanaka et al. / Biochimica et Biophysica Acta 1253 (1995) 9-12

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