Rat pancreatic lipase and two related proteins: enzymatic properties and mRNA expression during development

Abstract

We report the cDNA sequences of rat colipase, rat pancreatic lipase (rPL), and a rat pancreatic lipase-related protein (rPLRP). Comparison to the human PLRP cDNA suggests that the isolated clone encodes rPLRP-2. Both cDNA and a third cDNA encoding rPLRP-1 are secreted from Sf9 cells infected with recombinant baculovirus. rPL and rPLRP-2 hydrolyze triolein, 8.0 and 4.4 mumol.min-1.microgram-1, respectively. They are inhibited by bile salts, and activity is restored by (pro)colipase. PLRP-1 has barely detectable activity against triolein, even with (pro)colipase present. The pattern of mRNA expression during development in the rat reveals that all mRNA are low in the fetal rat pancreas. Both PLRP mRNA rise just before birth to a maximum 12 h after birth. They fall to low levels in the adult. In contrast, the PL mRNA is low at birth and rises rapidly during the suckling-weanling transition. In conclusion, the rat has at least three genes encoding different lipases, and these related genes have separate regulatory controls.

Fig. 1

The region amplified by polymerase chain reaction (PCR): nucleotide sequence for the region of rat pancreatic lipase (rPL), rat pancreatic lipase-related protein-1 (rPLRP-1) (18), and mouse PLRP-2 (mPLRP-2) (2, 3) amplified by PCR. Top sequence is for mPLRP2, a cDNA originally isolated from interleukin-4-stimulated, mouse cytotoxic T lymphocytes (3). Middle sequence is for rPLRP-1; bottom sequence is for rPL. Numbers refer to the base-pair position in the full-length sequence. Sequence of the oligonucleotide primers for the first PCR is underlined. Dashed double line marks nested primers for the second PCR. The 3′-primers were complementary to the given sequence.

Fig. 2

rPLRP-2 cDNA sequence and predicted protein sequence. Top line is nucleotide sequence, with untranslated region in lower-case letters and open reading frame in upper-case letters. The predicted amino acid sequence is given below in the single-letter amino acid code. Asterisk marks the stop codon. Putative active-site residues are in boldface; potential NH₂-glycosylation site is underlined.

Fig. 3

Nucleotide sequences and predicted amino acid sequences for rat procolipase and rPL. A: complete nucleotide sequence and predicted amino acid sequence for the lipase clone. B: the same information for the colipase clone. Top line of each panel shows the nucleotide sequence, with untranslated regions in lowercase letters and coding region in capital letters. The predicted amino acid sequence is given in the single-letter code below the nucleotide sequence.

Fig. 4

Expression of human PL (hPL), rPL, and rPLRP-2 in baculovirus-infected Sf9 cells. Aliquots (20 µl) of media from infected cells were collected 4 days postinfection and analyzed by SDS-PAGE and immunoblot with a polyclonal antibody against hPL. First lane, rPL; second lane, rPLRP-1; last lane, rPLRP-2.

Fig. 5

Activity of rPL and rPLRP-2 against triolein. Each protein (100 ng) was assayed in 50-µl volumes with the standard assay system (10). A: time course of the reaction in presence of deoxycholic acid (DOC) with and without human procolipase. B: inhibition of rPLRP-2 by various concentrations of DOC with and without human procolipase. Assay was done with 2.5% gum arabic; FA, fatty acid.

Fig. 6

Nucleotide sequence of the specific probes for PL and PLRP-1. A: sequence of the PL probe compared with the homologous region of PLRP-1 and PLRP-2. Double dots show bases that match the PL sequence. Calculated median temperature (T_m) of PL is 10°C for PLRP-1 and 17°C for PLRP-2 (14). Calculated T_m of PL for hybridization to itself is 73°C. B: PLRP-1 probe sequence compared with homologous sequence in PL and PLRP-2. Double dots show bases that are identical. Calculated T_m of PL is 20°C for PLRP-1, 17°C for PLRP-2, and 79°C for itself.

Fig. 7

RNA blots of rat pancreas RNA hybridized with specific probes. A: 3 slot blots containing cDNA for rPL, rPLRP-1, and rPLRP-2 hybridized with the specific probes described in Fig. 6. One hundred nanograms of each cDNA insert restricted from pGEM with EcoR I was denatured with 0.2 M NaOH and applied to a slot-blot apparatus containing a Hy-Bond N⁺ membrane. DNA was cross-linked to the membrane with ultraviolet light and hybridized to the probe indicated in the figure under conditions for dot blot given in METHODS. B: RNA blots of rat pancreas total RNA hybridized with each specific probe and an 18S RNA probe. RNA (25 µg) was separated on an agarose gel in formaldehyde (14) and transferred to Hy-Bond N⁺ membranes. Hybridization conditions were identical to those described in METHODS. Rat age is given above each line. B, 12 h after birth; 28, 28 days of age; A, adult; L, adult liver. Probe is given under each autoradiograph.

Fig. 8

Developmental patterns of colipase, PL, PLRP-1, and PLRP-2 mRNA in the rat. Dot blots containing RNA isolated from rat pancreas at various embryonic and postnatal ages were hybridized to oligonucleotide probes as described in METHODS. Relative mRNA levels are given at each age. A: results for PL. B: results for PLRP-1. C: results for PLRP-2. D: results for colipase. The birth, 0.5-day, 28-day, and adult points are averages of 3 separate determinations. All others are averages of 2 determinations.