Sphingolipid hydrolase activator proteins and their precursors

Vol. 165, No. 3, 1989 December

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

29, 1989

Pages

Sphingolipid

Hydrolase

Akira

Sano,

Takashi

Shu-ichi

The Department

Activator

of Neuropsychiatry,

*The Department

Proteins and Their Precursors

Hineno,

Tatsuo

Yasuo

Kakimoto,

Ueno,

1191-1197

Mizuno, and

Keiji Koji

Kondoh,

Inui*

Ehime University School of Medicine, Shigenobu, Onsen-gun, Ehime 791-02, Japan

of Pediatrics, Osaka University School of Medicine, Fukushima, Osaka 553, Japan

Received

October

30,

1989

Activator proteins for sphingolipid hydrolases (saposins) are small acidic, heat-stable glycoproteins that stimulate the hydrolysis of sphingolipids by lysosomal enzymes. The molecular mass of each stimulator is about 10 kDa, but glycosylated forms of higher mass exist too. The distribution and developmental changes in two saposins and their precursor proteins were studied with the aid of monospecific antibodies against saposin-B and saposin-C. They show a wide distribution in rat organs and forms intermediate between saposin and prosaposin (the precursor protein containing four different saposin units) could be seen. The amount of saposin and the degree of processing from prosaposin are quite different in different tissues. The saposins are the dominant forms in spleen, lung, liver, and kidney, while skeletal muscle, heart, and brain contain mainly precursor forms. In human blood, leukocytes contain mainly saposin, while plasma contains mainly precursor forms and platelets show many forms. Their subcellular distribution was studied using rat liver. The saposins of approximately 20 kDa are dominant in the light mitochondrial, mitochondrial, and microsomal fractions, following the distribution of the activity of a lysosomal marker enzyme. The nuclear fraction exhibits bands corresponding to non-glycosylated saposin. The soluble fraction contained much precursor forms. A developmental study of rat brain showed that the concentration of saposin precursors increased with age. 0 1989 AcademicPress, IrIG.

Recently, relatively

small, heat-stable, acidic glycoproteins

hydrolases acting on sphingolipids originally

that stimulate the action of lysosomal

have been described. The existence of some of these proteins was

described by Mehl and Jatzkewitz

(I), Ho and O’Brien (2), Li and Li (3), and Hechtman (4). They

seem to exert a relatively specific interaction with the enzymes or with the substrates, or both and, in some cases, require an acidic lipid for effectiveness. Some of the saposins occur in carbohydrate-free as forms bearing different oligosaccharides; action (5,6). They have been variously

form, as well

the carbohydrate moiety is not essential for the stimulatory

called saposins, cohydrolases, heat-stable factors, sphingolipid

activator proteins, etc. The term saposin (67)

is used here, abbreviated SAI? For recent reviews see ref. 8 and

9.

Abbreviations:

SAC saposin; SDS-PAGE, sodium dodecyl sulfate polyacrylamide

gel electrophoresis. 0006-291x/89

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Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 165, No. 3, 1989

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More recently it became clear that the saposins are derived by specific proteolytic

cleavage from a

precursor protein, called prosaposin, which consists of four very similar domains, each of which has saposin activity (10-14). Each of the four saposins consists of about 80 amino acids, including six similarly spaced cysteine residues in oxidised form, a glycosylation

site, and specifically located tyrosine and proline

residues. SAP-B is the stimulator of sulfatide, G M1 ganglioside hydrolys’is (15-17). SAPS-C stimulate

and, possibly, trihexosylceramide

the hydrolysis of glucosylceramide

This paper describes the presence of prosaposin and intermediates human blood. Striking differences in distribution

DEAE-Sephacel,

in its proteolysis in rat tissues and

of the different peptides were observed.

MATERIALS Materials-Bovine

and galactosylceramide.

AND METHODS

spleens were obtained from a slaughter house and stored at -80°C before use.

Sephadex G75 (fine), and CNBr-activated

Sepharose were from Pharmacia; acetonitrile,

HPLC grade, acrylamide, sodium dodecyl sulfate, and peroxidase conjugated anti-rabbit Wako Chemical; trifluoroacetic Immobilon

IgG were from

acid, HPLC grade was from Pierce; Freund’s adjuvants were from Difco;

transfer membranes (polyvinylidene

fluoride membranes) were from Millipore.

Preparation of saposin-C from bovine spleen and antibody against SAP-C-The

isolation procedure of

SAP-C was generally the same as the method described by Sano et al. (12). A hot-water spleen was purified by ammonium

sulfate precipitation,

DEAE-Sephacel

extract from bovine

ion exchange, Sephadex G-75 gel

permeation, and reverse phase HPLC with a C, column (Con A-Sepharose column chromatography omitted.). Amino acid sequencing and amino acid compositional significant

contamination

analysis of this preparation

was

showed no

with other proteins.’ Each of two young male rabbits was injected subcutaneously

with 100 pg of purified bovine SAP-C in Freund’s incomplete adjuvant. After 17 days another 100 pg in Freund’s incomplete adjuvant was injected subcutaneously. By four weeks after the initial injection, the plasma produced precipitin

lines in the Ouchterlony

double immunodiffusion

procedure. Two weeks after the

second injection blood was collected and an IgG fraction was isolated using ammonium sulfate precipitation and DEAE-Sephacel

column chromatography.

affinity chromatography,

The antibody was further purified to be monospeciflc by

using bovine SAP-C attached to CNBr-activated

was used in all experiments. Monospecific anti-human Tests with immunoblot

procedure showed there were no crossreactivity between our different monospecific

antibodies and antigens. The anti-human C, and the anti-bovine

SAP-B Ig G was reactive neither to pure guinea pig nor bovine SAP-

SAP-C Ig G was reactive neither to partially purified guinea pig nor bovine SAP-B.

SDS-PAGE and immunoblots-SDS-PAGE acrylamide (18). After electrophoresis detected indirectly

Sepharose. This preparation

SAP-B IgG was prepared as previously described (15).

was performed by the method of Laemmfi with 15%

the samples were electroblotted

to Immobilon

with the antibodies described above and peroxidase-conjugated

membranes and anti-rabbit

IgG and with

a peroxidase reactant, diaminobenzidine. RESULTS Organ distribution

of saposins

and

their precursors-An

anesthetized Wistar male rat was perfused

well with saline from the left ventricle of the heart after cutting femoral veins. Organs were taken out

lA. Sano, T. Mizuno, Z Hineno, K Kondoh, S. Ueno, Y Kakimoto, and N. Morita, unpublished 1192

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m Organ distribution of sqosins and their precursor form. Organ extracts (50 M of protein) were electrophoresed by SDS-PAGE, then immunoblotted as described in the text. 1A: detected with anti-SAP-B IgG, 1B: detected with anti-SAP-C. The arrow heads on the right sides are the positions of the molecular weight standards.

quickly and homogenized

in five volumes of iced 10 mM Na-phosphate,

pH 7.0, and centrifuged at 10,000 x g

for 10 min. The resultant supernatants were used as samples for SDS-PAGE. While immunoreactive

bands

could be seen with whole homogenates, the amount of tissue needed to get good reactivity was so large that the bands were distorted. The 10,000 x g supematant produced satisfactory bands and was therefore used for most of this study Immunoreactive

bands of a wide range in molecular weights were seen with the antibodies to both

SAP-B and SAP-C in all organs examined (Fig. I). The patterns of the bands were quite similar with both and kidney, while precursor

antibodies. The smaller forms were markedly dominant in spleen, lung, liver, forms were the major forms in skeletal muscle, heart, and brain.

The cellular components and plasma of human blood were separated by centrifugation

and washed

well with isotonic buffer, then examined as above (Fig. 2). Leukocytes contained a high level of saposins. Plasma contained precursor forms exclusively. Platelets showed many bands corresponding intermediate

precursor polypeptides. Red cells showed a single band corresponding

anhydrase, which reacts with diaminobenzidine Subcellular distribution homogenized

of sapsins

the position of carbonic

in peroxidase reaction.

and their precursor

in rut

in sucrose solution and fractionated by differential

liuer-Rat

liver perfused as above was

centrifugation.

Each fraction was assayed

for marker substances and enzymes (19). As shown in Fig. 3, a relatively high concentration forms was found in the nuclear fraction (N). In this experiment, the relative specific activities of the mitochondrial, showed the expected distribution

to prosaposin and

DNA

was

of the smaller

concentrated in the N fraction, and

lysosomal, microsomal,

and cytosolic marker enzymes

patterns (Fig. 4). The intensities of the saposin bands in the the nuclear

fraction increased when the particles were further purified according to the method of Chauveau et al.(ZO) (data not shown). 1193

Vol. 165, No. 3, 1989

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-3

B

A

;

NMLPS

m Distribution of immunoblotting were with anti-SAP-B IgG, the molecular weight

N

ML

PS

saposins and their precursor forms in human blood. Electrophoresis and performed as in Fig. I. The amount of samples were each 50 ccg of protein. 2A: detected 28: detected with anti-SAP-C. The arrow heads on the right sides are the positions of standards.

F&J. Subcellular distribution of saposins and their pwcursor forms in rut liver. Each 50 pg protein of the fractions was analysed. 3A: detected with anti-SAP-B IgG, 3B: detected with anti-SAP-C. The abbreviation above each lane is defined as follows: N, nuclear fraction; M, mitochondrial fraction; L, light mitochondrial fraction (lysosomal fraction); c microsomal fraction; S; cytosol.

Dezwlopfnentul changes in saposins and their precursors in raf brain-As

shown in Fig. 5, the saposins

and the precursor forms were already seen at birth. The concentrations of the precursor forms increased gradually from the 10th day after birth up to the last age point tested, 50 days.

N

M

Subcellular

L

P

Fractions

s

n q

DNA Cytochrome Oxidase Acid Phosphatase Glucose-6-phosphatase IJM

Fi11.4 Subcellular distribution of DNA and marker enzymes. The relative concentrations of DNA and relative specific activities of marker enzymes in the subcellular fractions (see legend for Fig. 3) are shown.

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494K

467K 443K 430K 420K 414K

4 6.5K

m Deoelo~nz,mental changes of saposins and their precursor forlns in rat brain. Brain extracts (50 gof protein) were analyzed. 5A: detected with anti-SAP-8 IgG, 58: detected with anti-SAP-C. The arrow heads on the right sides indicate the molecular weight standards.

DISCUSSION The many bands for the different saposins obtained by SDS-PAGE are mostly the result of carbohydrate heterogeneity

in the side chains as we demonstrated

the existence of prosaposin and its intermediates the length microheterogeneity demonstrated

of the polypeptide

in proteolytic

in the case of pure SAP-C (5), as well as

conversion, and are partially the result of

chains such as the N-terminal

microheterogeneity

in SAP-C (11),(12). According to the sequence analysis of cDNA for prosaposin (13),(14), it has

five N-glycosylation

sites in it. The four saposins occur in a single chain in prosaposin in the sequence A-B-C-

D (6), and one could expect that an antibody reactive to SAP-C would visualize trisaposins ABC and BCD and disaposins BC and CD. The immunoblot

results obtained with the preimmune serum showed no

significant bands, and the antibodies in this study are highly specific. It is interesting

that the antibodies

are reactive to the antigens of the different spieces but not to the different SAPS in spite of the structural similarity. The small saposins (-6 kDa) that are seen in submandibular

gland might be the dialyzable

activator

of glucosidase reported to occur in saliva (21). Its reactivity with antibodies (Fig. 1) indicates that it is structurally very is evidently

similar to the other saposins but lacks a significant portion of the usual form. This portion

unessential for stimulatory

activity.

Analysis of human blood revealed that plasma contains a considerable amount of prosaposin. Since plasma is a typical extracellular

fluid, it is likely that the precursor protein is secreted by certain cells into

the extracellular spaces. Newly formed molecules of sulfated glycoprotein, SGP-1, were found to be largely secreted into the cell medium by Sertoli cells (22) while human fibroblasts were found to process most of the newly formed saposin by proteolysis (23). Some kinds of leukocytes have the function of phagocytosis and have well differentiated

lysosomes, so it is reasonable to see much mature saposins there (Fig. 2). Spleen,

which also has hydrolysis as a major function, also has most of its saposin in the active, monomeric form (Fig. 1). It is possible that the precursor forms serve in other tissues as storage forms, to be rapidly converted to the active form when needed for a surge in hydrolytic workload. 1195

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In the developmental differentiation

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growth of rat brain , neuronal division is largely prenatal, and neuronal

such as synaptogenesis occurs mostly during the first 2 to 3 weeks of postnatal life. A peak in

the level of saposin appears at -15 days, an age at which the levels of ceramidase, sulfatase, and Bglucosidase are maximal in mouse brain (24). However, the ratio of precursor to mature form increases during rat brain development. Several species of prosaposin appear at all ages, increasing gradually

after day 10

(Fig. 4). This pattern of increase correlates with the steady rise seen in rat brain sphingolipids detected prosaposin in human cerebrospinal A number of biological

and pathological

fluid.2 functions are attributed

expression on cell surfaces has been correlated with cell transformation, and tumor progression. Glycolipids

also serve as differentiation

to different sphingolipids

markers and are thought to participate

hydrolases activator. If prosaposin or SGP-I has such a biological

extracellular

in

the binding of exogenous

factors, and cell recognition. It is not known yet whether prosaposin or SGP-1 has a biological sphingolipid

(26). Their

adhesion of cells to matrix proteins,

various cell regulatory functions, such as cell contact response, contact inhibition,

possibility that it controls building

(25). We also

activity as a

activity, there is a

and scrapping the sugar moieties of membranous

sphingolipids

from the

space.

Our finding that nuclei contain a relatively high level of SAPS-B and -C raises the possibility these proteins exert a modulatory

influence on the gene regulation

and cell proliferation.

that

The level of SAEs-

A, -C, and -D are very high in the spleens of Gaucher patients (6), which are distinctive for their greatly increased size and accumulation of glucosylceramide. glucosylceramide

together with an inhibitor

This condition can be mimicked by injecting mice with

of glucosidase; the level of SAP-C rises rapidly (27), as does

the size and DNA content of the liver (28).

Acknowledgments -This study was partly supported by Grant 01770180 from the Ministry of Education, Science and Culture, Japan, and by ON0 Medical Research Foundation (A. S.). We thank Dr. N. S. Radin of the Mental Health Research Institute, University of Michigan, U. S. A., for reviewing this manuscript.

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