©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characterization of an Apical Early Endosomal Glycoprotein from Developing Rat Intestinal Epithelial Cells (*)

(Received for publication, August 25, 1994; and in revised form, November 9, 1994)

Becky A. Speelman (1) Katrina Allen (1) Tamara L. Grounds (1) Marian R. Neutra (2) Tomas Kirchhausen (3) Jean M. Wilson (1)(§)

From the  (1)Departments of Cell Biology and Anatomy and of Pediatrics, Steele Memorial Children's Research Center, University of Arizona, Tucson, Arizona 85724, the (2)Gastrointestinal Cell Biology Research Laboratory, Children's Hospital, Boston, Massachusetts 02115, and the (3)Department of Cell Biology, Harvard Medical School and the Center for Blood Research, Boston, Massachusetts 02135

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The apical endosomal compartment is thought to be involved in the sorting and selective transport of receptors and ligands across polarized epithelia. To learn about the protein components of this compartment, we have isolated and sequenced a cDNA that encodes a glycoprotein that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. The deduced amino acid sequence predicts a protein of 1216 amino acids with a molecular mass of 133,769 Da. The deduced amino acid sequence together with aminoterminal amino acid sequencing indicate that there is a cleaved 21-amino acid signal sequence at the NH(2)-terminal portion of the molecule. There is a single hydrophobic region near the carboxyl terminus that has the characteristics of a membrane-spanning domain and a 36-amino acid cytoplasmic tail. We have found that the major form of this protein in intestinal epithelial cells has a molecular mass of 55-60 kDa, which is significantly smaller than the size predicted from the cDNA sequence, suggesting that the protein is synthesized as a large precursor and processed to the smaller form. The smaller form remains associated with the membrane, however, possibly through noncovalent association with the transmembrane portion of the molecule or with another membrane protein. The extracytoplasmic domain is cysteine-rich, with three cysteine-rich repeats that are similar to cysteine repeats present in several receptor proteins. However, there is no other significant similarity to other proteins in the GenBank. The cytoplasmic tail contains a possible internalization motif and several consensus motifs for serine/threonine kinases. Northern blot analysis suggests a single abundant message, and Southern blot analysis is consistent with a single gene and the absence of pseudogenes for this unique endosomal protein.


INTRODUCTION

After endocytosis from the plasma membrane, internalized receptors and ligands are delivered to endosomes (for review, see Trowbridge et al.(1993)). The endosomal compartment performs a variety of functions, including the sorting of internalized receptors and ligands (Abrahamson and Rodewald, 1981; Geuze et al., 1984, 1987) and newly synthesized lysosomal membrane proteins and hydrolases (Griffiths et al., 1988; Kornfeld and Mellman, 1989). In polarized epithelial cells, the apical endosomal compartment has been implicated in both apical to basolateral and basolateral to apical transepithelial transport (Abrahamson and Rodewald, 1981; Apodaca et al., 1994).

We previously generated a monoclonal antibody against a glycoprotein that is enriched in the apical tubular endosomes of absorptive cells in developing rat ileum (Wilson et al., 1987). On Western blots, this antibody recognizes a major band with an apparent molecular mass of 55-60 kDa and a minor high molecular mass band of 130 kDa. This endosomal antigen is expressed in high amounts during the time when ileal epithelial cells are actively endocytic, and expression stops when the intestinal epithelium achieves the adult configuration (Wilson et al., 1991). However, unlike the neonatal jejunum, the ileum is not involved in the transfer of IgG across the intestine (Rodewald, 1980) and instead has been suggested to be active in the uptake and transepithelial transport of milk-borne peptides and growth factors (Siminoski et al., 1986; Gonnella et al., 1987). To further study the composition of this endosomal compartment, we have isolated the cDNA for this endosomal protein and have characterized this protein at the molecular level. Analysis of the cDNA sequence suggests that this protein is synthesized as a high molecular weight precursor and post-translationally processed to lower molecular weight forms. Screenings of the DNA and protein data bases indicate there is no significant identity to other proteins in the GenBank and that it is a unique apical endosomal protein.


EXPERIMENTAL PROCEDURES

Isolation of Neonatal Rat Ileum Endosomal Antigen

Epithelial cells were isolated using the inverted loop method as described previously (Wilson et al., 1987). For each antigen isolation, 10-15-day-old neonatal rats were used. Enterocytes were homogenized in buffer containing 10 mM HEPES, pH 7.2, 0.25 M sucrose with 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml each of aprotinin and leupeptin (unless otherwise noted, all reagents came from Sigma). All solutions contained the same protease inhibitor mixture. The homogenate was spun at 100,000 times g for 1 h to yield a crude membrane fraction. Endosomal antigen isolation was carried out by the method of Bartles et al.(1985). Briefly, the crude membrane pellet was resuspended in buffer containing 0.5% Nonidet P-40, 0.2 M sucrose, 60 mM NaCl, 10 mM Tris-HCl pH 7.4, and then respun at 100,000 times g for 1 h. The supernatant was pumped through a mouse IgG column coupled to Sepharose 4B (Bio-Rad) followed by an anti-endosomal antigen monoclonal antibody column coupled to Affi-Gel 10 (Bio-Rad; 1.5-ml bead volume). The column was washed and eluted as described (Bartles et al., 1985). Fractions containing the endosomal antigen were concentrated by trichloroacetic acid precipitation. Western blots confirmed that the purified antigen cross-reacted with the monoclonal antibody.

Amino Acid Sequencing

For NH(2)-terminal sequencing, purified endosomal antigen was separated by SDS-PAGE (^1)(Laemmli, 1970) and electroblotted to polyvinylidene difluoride membrane (Bio-Rad) (LeGendre and Matsudaira, 1989). The 55-60-kDa band was excised from the blot and sequenced by automated Edman degradation. For internal amino acid sequencing, the immunopurified protein was separated by SDS-PAGE, and the band was excised from the gel, electroeluted for 3 h at 8 mA, and concentrated by trichloroacetic acid precipitation (Stone et al., 1989). For trypsin digestion (Stone et al., 1989), 5 µg of electroeluted protein was resuspended in 8 M urea, 0.4 M NH(4)HCO(3), 4.5 mM dithiothreitol and incubated with 10 mM iodoacetamide for 15 min at 25 °C. 0.5 µg of sequencing-grade trypsin (Boehringer Mannheim; 1:10, w/w) was added and incubated for 24 h at 37 °C. The reaction was separated on a Vydac C(18) reverse-phase column (2.1 mm, inner diameter, times 25 cm; The Separations Group, Hesperia, CA). Four peptide fragments were chosen for sequencing. Amino acid sequencing was performed at the University of Arizona Macromolecular Structures Facility using an Applied Biosystems 477A protein sequencer.

Cloning of the Endosomal Antigen cDNA

Amino-terminal and internal peptide sequences (see Fig. 2A and Fig. 3, diagonalunderlining) were used to design degenerate primers for reverse transcription followed by polymerase chain reaction. Primers were designed with inosine triphosphate in positions of highest degeneracy and mixed oligonucleotides at positions of lower degeneracy. EcoRI restriction sites were placed on the 5`-ends of the primer to facilitate subcloning. Primers were used after synthesis without purification. 19 µg of total neonatal rat ileum RNA was reverse-transcribed with 400 ng of internal antisense primer, 45 units of avian myeloblastosis reverse transcriptase (Boehringer Mannheim) for 1 h at 42 °C followed by ethanol precipitation (Frohman et al., 1988). The polymerase chain reaction was performed on the entire reverse transcription reaction with 3.5 µg of amino-terminal and internal primers. The reaction was premelted for 5 min at 95 °C; followed by 30 cycles for 1 min at 95 °C, 3 min at 42 °C, and 2 min at 72 °C; followed by a final extension for 10 min at 72 °C. The resulting single band of 440 bp was isolated, digested with EcoRI, subcloned into Bluescript KS vector (Stratagene Cloning Systems, La Jolla, CA), and sequenced using dideoxynucleotide sequencing (Sequenase, U.S. Biochemical Corp.) with universal and reverse primers. Additional clones were isolated by screening a neonatal rat intestine ZAP II library (Stratagene) by colony hybridization with the 440-bp fragment and individual plasmids obtained by phage rescue. DNA and protein data base screening was performed using the Wisconsin Genetics Computer Group sequence analysis software using BLASTA and BLASTX analysis.


Figure 2: Reverse transcription and polymerase chain reaction of total RNA from neonatal rat ileal epithelial cells. A, amino-terminal and internal amino acid sequences were used to design primers. Primers were designed with a 5`-GG clamp followed by an EcoRI restriction site (underlined) to facilitate subcloning into sequencing vectors. Deoxyinosine was used in positions of highest degeneracy and mixed oligonucleotides in positions of lower degeneracy. B, reverse transcription of total RNA using the internal primer followed by polymerase chain reaction using both primers resulted in a single band of 450 bp.




Figure 3: Nucleotide sequence and predicted amino acid sequence of the endosomal antigen cDNA. Nucleotides are numbered 5` to 3`, with the first nucleotide of the putative signal sequence at position 1. Amino acids are numbered from the first amino acid obtained by amino-terminal sequencing. The positions of the primers used for polymerase chain reaction are denoted by diagonalunderlining. The signal sequence is boxed, and the transmembrane domain is shown in a black box. The cysteine residues are indicated by shadedcircles, and consensus signals for N-linked glycosylation are enclosed in ovals.



Northern Blotting

Total RNA was obtained from isolated intestinal epithelial cells as described (Sambrook et al., 1989). 10 µg of total RNA was separated on a 1% formaldehyde-agarose gel, transferred to nitrocellulose (Bio-Rad), and hybridized according to published methods (Sambrook et al., 1989) at 68 °C with a 500-bp fragment from the 5`-end of the open reading frame. Labeling of the probe was accomplished by random prime labeling (Boehringer Mannheim) with [P]dATP. The blot was exposed to Kodak XAR-5 film at room temperature for 2 h.

Southern Blotting

20 µg of genomic rat liver DNA isolated as described (Sambrook et al., 1989) was digested for 2 h at 37 °C with 100 units of EcoRI, HindIII, NdeI, ScaI, and XbaI. After digestion, samples were electrophoresed on a 0.7% agarose gel, transferred to Hybond N (Amersham Corp.), and hybridized with a 100-bp fragment derived from nucleotides 395-495. Blots were exposed to Kodak XAR-5 film for 1 week at -80 °C with intensifying screens.

In Vitro Transcription and Translation

8 µg of BKS plasmid containing the complete open reading frame was linearized with SalI and transcribed using T3 DNA polymerase (Stratagene). Translation was carried out in the presence of TranS-label (ICN Radiochemicals, Irvine, CA) in a rabbit reticulocyte lysate (Stratagene). Control translations were carried out in the absence of RNA. The reaction was separated by SDS-PAGE and exposed to Kodak XAR-5 film.


RESULTS AND DISCUSSION

Protein Sequencing

Because the major band isolated by immunoaffinity purification was the 55-60-kDa band (Fig. 1), this band was used for amino-terminal and internal amino acid sequencing. In the case of amino-terminal sequencing, although the amino terminus was not blocked, only 6 amino acids were obtained in each of three attempts (Table 1). For internal amino acid sequencing, immunopurified endosomal antigen was trypsin-digested, fragments were separated by HPLC, and selected fragments were sequenced by Edman degradation. Four internal amino acid sequences were obtained and are shown in Table 1.


Figure 1: Purified endosomal antigen. The endosomal antigen immunopurified from neonatal rat ileal epithelial cells was separated on a 7.5% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane. The 55-60-kDa band (arrow) was excised from the membrane for amino-terminal sequencing or was electroeluted and digested with trypsin for internal amino acid sequencing.





Cloning and Sequencing of the Endosomal Antigen cDNA

After unsuccessful screenings of a neonatal rat intestine cDNA library with mixed oligonucleotide probes, an initial cDNA fragment for the endosomal antigen was identified by polymerase chain reaction using degenerate oligonucleotide primers. As shown in Fig. 2, primers derived from the amino-terminal amino acid sequence and internal amino acid sequence 1 were designed with deoxyinosine at positions of highest degeneracy and mixed oligonucleotides at positions of lower degeneracy. Reverse transcription using the internal primer followed by polymerase chain reaction resulted in a fragment of 450 bp (Fig. 2) that was subcloned into Bluescript vector for sequencing. Sequencing of this fragment resulted in the identification of a stretch of peptide sequence that had been independently obtained by sequencing of peptide fragments (Table 1), thus confirming that the cDNA identified was a fragment of the cDNA encoding for the endosomal antigen.

A ZAP cDNA library derived from neonatal rat intestine was then screened by colony hybridization with this 450-bp fragment, and larger clones were identified. The largest clone was 3.9 kb in length. Three independent clones of 3-3.7 kb were identified, and they were shown to be included within the sequence of the largest clone, indicating that the large clone was not due to artifactual joining of smaller clones. The full-length coding sequence was obtained by a combination of primer extension sequencing and nuclease deletion in both directions, and the open reading frame was determined. Additional peptide sequences obtained from internal amino acid sequencing were identified in the larger clones. The largest clone identified was not as large as the mRNA seen on Northern blots (see below) and did not contain a poly(A) tail. However, several screenings of the ZAP library with probes derived from the 3`-end of the clone did not result in larger clones or the identification of a poly(A) stretch. 3`-Rapid amplification of cDNA ends with primers derived from the 3`-end of the coding sequence did result in the identification of a fragment of 750 bp that terminated in a poly(A) tail (data not shown).

The cDNA sequence of this clone and the predicted amino acid sequence are shown in Fig. 3. The cDNA contains 57 bases of 5`-untranslated region, and the first methionine in the amino acid sequence contains a purine in position -3, an important component of the consensus motif for an initiation methionine (Kozak, 1989). The next 21 amino acids are quite hydrophobic and probably encode a signal peptide followed by the 6 amino acids obtained from NH(2)-terminal peptide sequencing. The putative signal cleavage site conforms to the -1,-3 rule, with a glycine in position -1 and a serine in position -3 (von Heijne, 1986). The open reading frame after the signal peptide encodes a protein of 1195 amino acids with a calculated molecular mass of 131,128 Da.

Because of the size of the predicted open reading frame and the presence of a high molecular weight band on immunoblots, we postulated that the protein is synthesized as a high molecular weight precursor and proteolytically processed into the smaller form seen on Western blots. To determine the size of the protein encoded by the cDNA, we performed in vitro transcription and translation of the 3.9-kb clone in a rabbit reticulocyte lysate. In vitro translation resulted in a single specific band of M(r) 130,000 (Fig. 4). Other forms were absent, suggesting that the processing event that results in the smaller in vivo form occurs post-translationally in the endoplasmic reticulum, Golgi apparatus, or on the cell surface. Even after cleavage, the smaller form remains associated with the membrane (Wilson et al., 1987) and may remain noncovalently associated with the transmembrane portion of the molecule. This arrangement would be similar to the brush-border glycosidases sucrase-isomaltase and lactase-phlorizin (Semenza, 1986). In the case of sucrase-isomaltase, pancreatic enzymes are thought to have a role in the post-translational processing to the mature form (Hauri et al., 1979). The mechanism and site of cleavage of the endosomal antigen remain unknown.


Figure 4: In vitro transcription and translation of the endosomal antigen cDNA. The 3.9-kb cDNA for the endosomal antigen was transcribed using T3 DNA polymerase and translated in a rabbit reticulocyte lysate. Lane1, translation in the absence of RNA; lane 2, translation in the presence of 1 µg of RNA. A single specific band of M(r) 130,000 is present.



In addition to the hydrophobic signal peptide at the amino-terminal portion of the protein, Goldman-Engelman-Steitz analysis (Engelman et al., 1986) with a window size of 20 indicates a single hydrophobic domain of 30 amino acids near the carboxyl terminus of the protein (Fig. 5). This hydrophobic domain is flanked by an arginine (position 1128) and lysine (position 1160) and is proposed to be a membrane-spanning domain. This predicted topology would indicate that this protein is a type I membrane protein, with the majority of the molecule on the extracytoplasmic face of the membrane. The extracytoplasmic domain is mostly hydrophilic and contains several cysteine-rich domains ( Fig. 3and Table 2). There are three cysteine-rich repeats in the protein at positions 7-32, 210-246, and 435-470 (Table 2). These repeats have similarity to cysteine repeats in a array of membrane proteins, including the alpha(2)-macroglobulin receptor (low density lipoprotein receptor-related protein) (Herz et al., 1988; Kristensen et al., 1990; Strickland et al., 1990), low density lipoprotein receptor (Goldstein et al., 1985), and members of the complement family (DiScipio et al., 1984) (Table 2). The functions of these repeats in these proteins are unclear. Overall, this endosomal antigen is relatively cysteine-rich, with 39 cysteine residues in the mature molecule. At least some of these cysteine residues are thought to be involved in disulfide bonds, as SDS-polyacrylamide gels run under nonreducing conditions result in an increased electrophoretic mobility of the protein. (^2)These intramolecular disulfide bonds may protect the protein from proteases present in the intestinal lumen or in the apical giant lysosome, a structure that is a morphological hallmark of neonatal rat ileal epithelial cells (Cornell and Padykula, 1969).


Figure 5: GES analysis of the predicted amino acid sequence. The deduced amino acid sequence was analyzed for the presence of nonpolar transbilayer helices employing the algorithm of Engelman et al.(1986). The window size is set for 20 residues. Two hydrophobic domains are identified, the amino-terminal signal sequence and a carboxyl-terminal domain that is proposed to be a transmembrane domain.





There are six consensus signals for asparagine-linked glycosylation in the extracytoplasmic domain (Fig. 3). Four of these signals are clustered toward the amino terminus of the protein with the asparagine residues at positions 183, 269, 320, and 346, and the remaining two are at positions 618 and 818. These consensus signals are located outside the cysteine-rich domains. Biochemical evidence indicates that at least three of these glycosylation sites are, in fact, glycosylated.^2

As mentioned above, near the carboxyl terminus of the predicted amino acid sequence, there is a hydrophobic stretch of 30 amino acids, beginning at amino acid 1128, that is characteristic of a membrane-spanning domain (Fig. 5). The portion of the molecule predicted to lie on the cytoplasmic side of the membrane is 36 amino acids in length. On the cytoplasmic side, separated from the membrane by 15 amino acids, is the amino acid sequence FDNILF(1174-1179), a sequence similar to the internalization signal FDNPVY found in the low density lipoprotein receptor (Chen et al., 1990). Although the majority of this endosomal antigen is present in early endosomal membranes, some immunoreactivity is detected on the apical cell surface of neonatal rat enterocytes (Wilson et al., 1987). An internalization motif may be necessary to allow cycling of this protein between the two membrane domains.

Some internalization motifs have also been shown to be related to the determinants involved in the basolateral targeting of receptors and membrane proteins (Brewer and Roth, 1991), and the low density lipoprotein receptor is normally targeted to the basolateral plasma membrane of epithelial cells. Interestingly, one of the basolateral targeting signals in the low density lipoprotein receptor includes the tyrosine (position 807 of the human receptor (Goldstein et al., 1985)) of the internalization motif, followed by a cluster of negatively charged amino acids (Matter et al., 1992). In the case of the endosomal antigen, which is present in the apical endosomal compartment, the presence of phenylalanine (amino acid 1179) in the analogous position in place of tyrosine may explain the apical endosomal targeting of this protein.

In addition to their roles in internalization and polarized targeting of membrane proteins, cytoplasmic domains are important for the sorting of membrane proteins to specific organelles. The determinants for the sorting of mannose 6-phosphate receptors to the late endosomal compartment have been shown to reside in two portions of the cytoplasmic domain, a tyrosine-containing internalization signal and a carboxylterminal dileucine motif (Jadot et al., 1992; Johnson and Kornfeld, 1992). Also, dileucine or leucine-isoleucine pairs have been implicated in the intracellular targeting and/or endocytosis of other proteins (Letourneur and Klausner, 1992; Pieters et al., 1993; Verhey and Birnbaum, 1994). The endosomal antigen described here has neither of these motifs, but does contain a isoleucine-leucine pair in the cytoplasmic tail.

Phosphorylation of the cytoplasmic tails of membrane proteins can also affect their intracellular trafficking (Casanova et al., 1990). The cytoplasmic domain of the endosomal antigen contains several serine and threonine residues, and there is a consensus motif for phosphorylation by casein kinase I (SXX(S/T), positions 1190-1193) and two motifs for phosphorylation by casein kinase II (TXXEX and SXXDX, positions 1186-1190 and 1172-1176, respectively) (Pearson and Kemp, 1991). However, it is not yet known whether this protein is phosphorylated. Determination of the role of the cytoplasmic domain in the endosomal targeting, retention, and endocytosis of this molecule awaits mutagenesis experiments.

Southern Blot Analysis of Rat Liver Genomic DNA

As described above, biochemical studies have indicated that there are several molecular weight forms of the endosomal antigen. To determine if the different forms of this protein arose from the transcription of multiple genes, we probed a blot of rat genomic DNA that had been digested with EcoRI, HindIII, NdeI, ScaI, and XbaI using a 100-bp fragment derived from the coding region. Under high stringency conditions, a single band was identified with each digest, indicating that there is only one gene for this endosomal antigen (Fig. 6A).


Figure 6: Southern and Northern blot analyses. A, Southern blot of rat genomic DNA. Genomic DNA was digested with enzymes as indicated, transferred to nitrocellulose, and hybridized with a 100-bp fragment spanning nucleotides 395-495 of the coding region. B, Northern blot of neonatal rat ileum total RNA. 3 µg of RNA was probed with a cDNA fragment derived from nucleotides 64-510. The message is 4.8 kb in length (arrow).



Northern Blot Analysis of Total Rat Ileum RNA

Northern blot analysis of total RNA derived from neonatal rat ileum enterocytes showed that there is an abundant message of 4.8 kb (Fig. 6B). These results may indicate that the different forms of this protein seen on Western blots are not due to alternative splicing of mRNA or transcription of multiple genes and are instead due to post-translational modification of the protein. However, due to the size and abundance of the message, small exon insertions or deletions would not have been detected on the Northern blot. Preliminary results from screening a cDNA library derived from hepatoma cells indicate that alternatively spliced forms of this protein may, in fact, exist. (^3)

This endosomal protein is a unique marker for a specialized apical endosomal compartment in intestinal epithelial cells. It will be of interest to determine the role of its structure upon its targeting and retention in the endosomal compartment of polarized cells. Also, as expression of this protein is correlated with the assembly of the apical endosomal complex in developing ileal cells (Wilson et al., 1991), it will be interesting to determine whether this protein plays a role in the extent or morphology of the endosomal compartment in other cell types.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK-43329 (to J. M. W.) and by a grant from the National Institutes of Health (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L37380[GenBank].

§
To whom correspondence should be addressed: Dept. of Cell Biology and of Anatomy, 1501 N. Campbell Ave., University of Arizona, Tucson, AZ 85724. Tel.: 602-626-2557; Fax: 602-626-2097.

(^1)
The abbreviations used are: PAGE, polyacylamide gel electrophoresis; bp, base pair(s); kb, kilobase pairs(s); HPLC, high pressure liquid chromatography.

(^2)
B. A. Speelman and J. M. Wilson, manuscript in preparation.

(^3)
B. A. Speelman and J. M. Wilson, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. Bryant Benson and Monica Chawla for assistance with the HPLC, Drs. Mary Rykowski and Ray Runyan for helpful discussions, and Drs. Paul St. John and James Casanova for comments on the manuscript.


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