Exp. Eye Res. (1992), 55, 645-647

LETTER TO THE EDITOR

N e w Members of the Lipocalin Family in Human Tear Fluid H u m a n and animal tear fluid is a very complex mixture whose proteic composition is now being extensively studied. Bonavida, Sapse and Sercarz (1969) described, for the first time, a protein present at high levels in tears of humans and several animal species (rabbit, rat, monkey). Its electrophoretic mobility was larger than albumin and it was absent from serum and other biological fluids such as cerebrospinal fluid, saliva, nasal secretion and sweat. This protein was called 'tear specific prealbumin': this is an inappropriate term since transthyretin is now the scientific name for prealbumin. Its local synthesis by lacrimal glands was shown by culturing lacrimal gland slices in the presence of radiolabefied amino-acid (Bonavida et al., 1969; ]anssen and Van Bijsterveld, 1980). Using higher resolution electrophoretic methods, we were able to demonstrate that Bonavida's team had not described a single protein but a group of at least six proteins whose molecular weight ranged from 15 to 20 kDa with isoelectric points ranging from 4.6 to 5.4 (Gachon et al., 1979; Gachon, Lambin and Dastugne, 1980); we called them 'proteins migrating faster than albumin' (PMFAs) in order to differentiate them from albumin or transthyretin. We also described protein G; specific tear protein with a badly chosen name that led to confusion of this protein with the G proteins involved in signal transduction. Its molecular weight was 3 1 0 0 0 and, after treatment with a reducing agent, it appeared to be composed of two subunits of similar molecular weight to PMFAs. The heterogeneity of these specific proteins was also demonstrated in rabbit tears (Gachon et al., 1986) and a protein susceptible to reducing agent was also reported. Anticipating a role for these proteins and in order to normalize the nomenclature, in this article we propose to call them 'tear lipocalin' (TL: followed by their molecular weight and their phi) and use the term 'tear lipocalin aggregate' (TLA: followed by the molecular weight in kDa of the aggregate) for TL association. Thus, protein G would be named TLA 31. During many years' work on tear specific proteins, it has become apparent that it is necessary to elucidate the complete amino acid sequence of these specific proteins since: (a) it is now possible (Doofittle, 1987) to model a putative protein if it is homologous with a protein of known structure, this would allow determination of their as yet unknown role; and (b) in eucaryotes most proteins which are transported to the extracefiular space are synthetized as precursor polypeptides containing cleavable N-terminal signal or targeting sequences. However, some proteins are exported from the cytosol without being proteolytically 0014-4835/92/100645 + 03 $08.00/0

processed (Tabe et al., 1984); furthermore, some secretory proteins were recently described (Rubartelli et al., 1990; Grundman et al,, 1986; Abraham et al., 1986; Ishikawa et al., 1989 ; Jaye et al., 1986) that do not possess hydrophobic signal sequences as stated both by examination of amino acid sequence and nucleotide sequence of the corresponding cDNA. So, it is possible that they follow a novel secretory route clearly distinct from the classical route, i.e. through the endoplasmic reticulum and Golgi apparatus. Consequently, the study of these tear-specific proteins and the sequence of their corresponding cDNA would permit better understanding of their mechanism of secretion and confirm whether they are synthetized as pre-proteins or not. Two-dimensional gel electrophoresis is generally considered the method with highest resolution for protein separation at the microgram level. Furthermore, it is possible to sequence protein spots after elution or electroblotting onto appropriate membranes such as polyvinylidene difluoride membranes (PVDF). Previous studies (unpublished data) and data from the literature (Fullard and Kissner, 1991) indicate that the same isoforms are present in all the subjects and that inter-subject differences in the isoforms distribution is very low. For those reasons, tears were collected from only one male donor. Proteins were separated by the procedure of O'Farrell (1975) as modified by Walsh et al. (1988) for avoidance of protein N-terminal blockage and tryptophan destruction. The transfer was performed according to Bauw et al. (1989) and PVDF membrane bound proteins were visualized by amidoblack staining. The sequence analysis was carried out with a gas phase sequencer (model 470 A, Applied Biosystem) equipped with an on-line phenylhydantoin amino acid derivative analyser. Table I shows NH 2 terminal amino acid sequence obtained for three h u m a n TL. Pairwise sequence comparison showed: (i) 82% identity in a 28 aminoacid overlap between TL 18/5'3 and TL 18/5.2; and (ii) 91% identity in a 23 amino-acid overlap between T L 1 8 / 5 . 2 and TL17/4.9. Amino acid sequence alignments indicate that TL 17/4"9 is shorter on the NH2 terminal side. Thus, it appears that these proteins resemble each other at a high degree in their Nterminal sequences. In Table II, homologies obtained from a protein data bank are given. Comparisons of the NH 2 terminal sequences of these proteins with all sequences in the NBRF protein data base revealed similarities to a group of 18-25 kDa proteins that belonged to a family of © 1992 Academic Press Limited

A. DELAIRE; E T A L .

646

TABLE I

N-terminal amino-acid sequences of TL 18/5"3, TL 18/5"2 and TL 17/4"9 (single letter code) TL 18/5'3" TL 18/5"2" TL 17/4"9" TL 18/5"3 TL 18/5'2

H R

S /

L L

L L

A A T T

S S S W W

D D D Y Y

E E E L L

D D

V V

S S

G G

TL 17/4.9

D

V

S

TL 18/5.3 TL 18/5-2 TL 17/4.9

T T T

V V V

D D D

E E E K K

I I I A A

G

T

W

Y

/ / /

E Tr E

F F F

P

L

K

A

E

M

N

P

E

M

N

Q Q Q M M M

* Note the presence, in each sequence, of the highly conserved stretch G-X-W-Y.In TL 18/5.2, Tp represents phosphate threonine. TABLE II

Alignment of the amino-acid sequence (single-letter code) of TL 18/5.3 with the sequences of members of the hydrophobic molecule transporter family TL18/5.3 VEG OBLG BG OBPr

H F V

S -T

L P Q

L T T

A

S

D T G

E E L

E E D

I N I

Q Q Q

D D K

M

K

P

P

V

M

K

E T A A S A

N V W S N D

L D D D C N

D -K I K V

I E E S Q E

G

L

E

E

N

K

--

S F I L F K

P P P L L V

S -D D Q A

E M K A M --

N K Q K E

L F S S G

-G A D G

V V V V V S S P N S

S S A T N

G G G G G

T T T V D

W W W W W

Y Y Y Y R

--S G T

L L L I L

K K A A Y

A A M A I

M

A A A V

VEG, Von Ebner's gland protein (Schmale, Holtgreve-Grezand Christiansen, 1990); OBLG,ovine fl lactoglobulin (Papiz et al., 1986); BG, Bowman's gland protein (Lee, Wells and Reed, 1987); OBPr, rat odorant binding protein (Pevsner et al., 1988). extracellular lipophilic-molecules carriers. Based on the three-dimensional structure of three proteins (retinol binding protein, fl-lactoglobulin and bilin binding protein) and on stretches of sequences which are strongly conserved, a new protein superfamily called the lipocalin family (Pervaiz and Brew, 1987) was suggested to be highly appropriate for binding a variety of hydrophobic ligands such as retinoids, steroids, bilins and lipids (Godovac-Zimmermann, 1988). In such a family, ligands are bound within the central cavity consisting of a barrel formed by two orthogonal g-sheets and an ~-helix. The putative role of the ~-helix is to induce protein aggregates, previously observed with ~ 1 microglobulin and several of the other lipocalins (Akerstr6m and L6gdberg, 1990). It was suggested that the ligand specificity was obtained by changing the residue types inside the central cavity. From recent studies (Schmale, Holtgreve-Grez and Christiansen, 1990) it appears that the most strongly conserved stretch of amino acids is the gly-X-trp-tyr part (X is an unspecified amino acid) of the first strand (A) of g-sheet. The functional importance of this conserved region is at present uncertain although, in fl-lactoglobulin and retinol binding globulin, the side chain of trp has been proposed to be part of the ligand binding site. Computer alignment o f amino acid sequences indicated significant homologies between TL 18/5.2, rat Von Ebner's gland protein (Schmale HoltgreveGrez and Christiansen, 1990), ovine fl-lactoglobulin

(Papiz et al., 1986), frog Bowman's gland protein (Lee, Wells and Reed, 1987) and rat olfactory binding protein (Pevsner et al., 1988). The TL shares the strongest conserved sequence, i.e. gly-X-trp-tyr, which is found in almost all the members of the family, in a section close to the amino terminus. Because of the similarity between the lacrimal and salivary tissues, the high level of homology between TL and VEG protein has to be emphasized: the Von Ebner's glands are small tubulo-alveolar salivary glands, located beneath the circumvallate and the foliate papillae; the VEG protein is a highly expressed secretory protein with a 18-kDa molecular weight. Our results agree in part with the short N-terminal sequences (ten residues) attributed recently to P~ and P4 proteins (Fullard and Kissner, 1991). These proteins were purified using high performance liquid chromatography and chromatofocusing fast protein liquid chromatography. Discrepancies between the sequence of TL 18/5.2 and protein P2 as between TL 17/4.9 and protein P~ may be attributed either to the previously described polymorphism of these proteins (Azen, 1976) or to the difficulty in interpreting chromatographic data of sequence analyses. It has been established that, in h u m a n plasma, transthyretin exists in a complex with retinol and retinol binding protein and that retinol is present in tears (Ubels and Mac Rae, 1984). Recently, the same group demonstrated convincingly that rabbit lacrimal gland produces RBP mRNA, and synthesizes and

LIPOC.,~LIN FAMILY PROTEINS IN HUMAN TEARS

secretes the corresponding protein (Ubels et al. 1991); RBP had previously been reported to be absent in h u m a n tears (Rask et al., 1980), but the availability of improved techniques m a y n o w allow confirmation of this point. Chao and Butala (1986) have isolated a TL (probably TL 18/5"3) and determined its amino acid composition: it is not certain if a specific complex is formed between [~H]retinol and this TL. Now, our aims are to: (i) k n o w the complete sequence of the TL; (ii) assess the relation of these proteins to the lipocalin family; and (iii) find w h i c h ligands interact specifically with TL.

Acknowledgements We thank B. Dastugue and all the members of the laboratory for helpful discussions. This work was supported by a grant from the French Department of Education. A. DELAIRE H. LASSAGNE A. M. F. GACHON*

Laboratoire de Biochimie Mgdicale, Facult~ de M~decine, 28, P/ace Henri Dunant, BP 38, 63001 Clermont-Ferrand Cede)(, France * For correspondence

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(Received Chicago 17 March 1992 and accepted in revised form 3 June 1992)