Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes

Abstract

Transcripts encoding a novel member of the lipoprotein receptor superfamily, termed LDL receptor-related protein (Lrp)13, were sequenced from striped bass (Morone saxatilis) and white perch (Morone americana) ovaries. Receptor proteins were purified from perch ovary membranes by protein-affinity chromatography employing an immobilized mixture of vitellogenins Aa and Ab. RT-PCR revealed lrp13 to be predominantly expressed in striped bass ovary, and in situ hybridization detected lrp13 transcripts in the ooplasm of early secondary growth oocytes. Quantitative RT-PCR confirmed peak lrp13 expression in the ovary during early secondary growth. Quantitative mass spectrometry revealed peak Lrp13 protein levels in striped bass ovary during late-vitellogenesis, and immunohistochemistry localized Lrp13 to the oolemma and zona radiata of vitellogenic oocytes. Previously unreported orthologs of lrp13 were identified in genome sequences of fishes, chicken (Gallus gallus), mouse (Mus musculus), and dog (Canis lupus familiaris). Zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) lrp13 loci are discrete and share genomic synteny. The Lrp13 appears to function as a vitellogenin receptor and may be an important mediator of yolk formation in fishes and other oviparous vertebrates. The presence of lrp13 orthologs in mammals suggests that this lipoprotein receptor is widely distributed among vertebrates, where it may generally play a role in lipoprotein metabolism.

Keywords: affinity purification; egg; endocytosis; low density lipoprotein receptor-related protein 13; oocyte; oogenesis; ovary; very low density lipoprotein receptor; vitellogenesis; yolk.

Fig. 1.

Left: ClustalW dendrogram showing relationships between low-density lipoprotein receptor family polypeptide sequences. GenBank accession numbers are provided. Numbers above each branch are pairwise- (p-)distances. Right: Models representing linear domain structures of LDLR family members as defined at the bottom.

Fig. 2.

Syntenic arrangements of zebrafish and Nile tilapia lrp13 and vldr (lr8) loci aligned to illustrate orthologies between the two species.

Fig. 3.

Left: Chromatograph of protein-affinity purified white perch Vtgrs. The dark bar indicates column pass through and transition to elution buffer. Right: Silver stained nonreducing 5% acrylamide gel of affinity purified Vtgrs. Numbers to the left indicate the sizes of molecular mass markers (kDa). Positions of VtgAa/b (Vtg) and ovary Vtgrs (arrows) are indicated to the right. The white arrow indicates a putative protein aggregate.

Fig. 4.

Left: Nonreducing 7.5% acrylamide gel stained with Coomassie brilliant blue (CBB) and ligand blot (LB) of white perch ovary membrane proteins. The LB in lane 2 was prepared using 0.25 μg/ml of DIG-labeled VtgAa/b and the LB in lane 3 was performed in the presence of a 200-fold excess molar ratio of unlabeled VtgAa/b. Right: Nonreducing 5% acrylamide gel Western blot (WB) of white perch ovary membrane proteins. The WB in lane 4 was prepared with α-WpLrp13 and the WB in lane 5 was performed with α-WpLr8−. Numbers to the left of gels or blots indicate the sizes of molecular mass markers (kDa). Positions of VtgAar, VtgAbr, pLDLR, Lr8−, and Lrp13 are indicated.

Fig. 5.

Semi-quantitative RT-PCR of lrp13 and rpl9 in striped bass tissues. The PCRs were terminated after 25, 30, and 35 cycles (indicated at the left).

Fig. 6.

Real-time quantitative RT-PCR of lrp13 and lr8− (left) and quantitative mass spectrometry of Lrp13 (right) in ESG, MVG, LVG, and PVG striped bass ovary. Vertical bars and data points represent mean values and brackets indicate standard errors of the mean. Mean values bearing the same letter superscript are not significantly different (P < 0.05 ANOVA and Tukey-Kramer HSD).

Fig. 7.

In situ hybridization of lrp13 in striped bass ovary. Hematoxylin-eosin stain (A, B) and adjacent sections (C, D) probed with antisense lrp13. ESG, MVG, and LVG oocytes are indicated in (A) and (B). Blue signal in (C) and (D) represents lrp13 localization.

Fig. 8.

Immunohistochemistry of Lrp13 in striped bass ovary. Phase contrast (A, B), fluorescence (C, D), and image overlays (E, F) of phase contrast and fluorescence. The boxed area in (F) is shown at high magnification in phase contrast (G) and fluorescence (H). ESG, MVG, and LVG oocytes are indicated in (A) and (B). Red signal in (C–F) and (H) represents Lrp13 localization and magenta signal in (H) represents DAPI-stained nuclei.